Molecules of the card-related protein family and uses thereof

ABSTRACT

Novel CARD- 9,  CARD- 10,  or CARD- 11  polypeptides, proteins, and nucleic acid molecules are disclosed. In addition to isolated CARD- 9,  CARD- 10,  or CARD- 11  proteins, the invention further provides CARD- 9,  CARD- 10,  or CARD- 11,  fusion proteins, antigenic peptides and anti-CARD- 9,  CARD- 10,  or CARD- 11  antibodies. The invention also provides CARD- 9,  CARD- 10,  or CARD- 11  nucleic acid molecules, recombinant expression vectors containing a nucleic acid molecule of the invention, host cells into which the expression vectors have been introduced and non-human transgenic animals in which a CARD- 9,  CARD- 10,  or CARD- 11  gene has been introduced or disrupted. Diagnostic, screening and therapeutic methods utilizing compositions of the invention are also provided.

CROSS REFERENCE TO RELATED APPLICATIONS

This application is a continuation of U.S. application Ser. No.09/798,412, filed Mar. 2, 2001, now abandoned, which is acontinuation-in-part of U.S. application Ser. No. 09/728,260, filed Dec.1, 2000, now abandoned, which is a continuation-in-part of U.S.application Ser. No. 09/685,791, filed Oct. 10, 2000, now abandoned,which is a continuation-in-part of U.S. application Ser. No. 09/513,904,filed Feb. 25, 2000, now abandoned, which is a continuation-in-part ofapplication Ser. No. 09/507,533, filed Feb. 18, 2000, which claimedpriority from provisional application Ser No. 60/168,780, filed Dec. 3,1999. The content of each of these applications is herein incorporatedby reference.

BACKGROUND OF THE INVENTION

In multicellular organisms, homeostasis is maintained by balancing therate of cell proliferation against the rate of cell death. Cellproliferation is influenced by numerous growth factors and theexpression of proto-oncogenes, which typically encourage progressionthrough the cell cycle. In contrast, numerous events, including theexpression of tumor suppresser genes, can lead to an arrest of cellularproliferation.

A particular type of cell death called apoptosis occurs indifferentiated cells when an internal suicide program is activated. Thisprogram can be initiated by a variety of external signals as well assignals that are generated within the cell in response to, for example,genetic damage. For many years, the magnitude of apoptotic cell deathwas not appreciated because the dying cells are quickly eliminated byphagocytes, without an inflammatory response.

The mechanisms that mediate apoptosis have been intensively studied.These mechanisms involve the activation of endogenous proteases, loss ofmitochondrial function, and structural changes such as disruption of thecytoskeleton, cell shrinkage, membrane blebbing, and nuclearcondensation due to degradation of DNA.

The various signals that trigger apoptosis are thought to bring aboutthese events by converging on a common cell death pathway, the corecomponents of which are highly conserved from worms, such as C. elegans,to humans. In fact, invertebrate model systems have been invaluabletools in identifying and characterizing the genes that controlapoptosis. Despite this conservation of certain core components,apoptotic signaling in mammals is much more complex than ininvertebrates. For example, in mammals there are multiple homologues ofthe core components in the cell death signaling pathway.

Caspases, a class of proteins central to the apoptotic program, areresponsible for the degradation of cellular proteins that leads to themorphological changes seen in cells undergoing apoptosis. Caspases arecysteine proteases having specificity for aspartate at the substratecleavage site. Generally, caspases are classified as either initiatorcaspases or effector caspases, both of which are zymogens that areactivated by proteolysis that generates an active species. An effectorcaspase is activated by an initiator caspase which cleaves the effectorcaspase. Initiator caspases are activated by an autoproteolyticmechanism that is often dependent upon oligomerization directed byassociation of the caspase with an adapter molecule.

Apoptotic signaling is dependent on protein-protein interactions. Atleast three different protein-protein interaction domains, the deathdomain, the death effector domain and the caspase recruitment domain(CARD), have been identified within proteins involved in apoptosis. Afourth protein-protein interaction domain, the death recruiting domain(DRD) was recently identified in murine FLASH (Imai et al. (1999) Nature398:777-85).

Many caspases and proteins that interact with caspases possess a CARDdomain. Hofmann et al. ((1997) TIBS 22:155) and others have postulatedthat certain apoptotic proteins bind to each other via their CARDdomains and that different subtypes of CARD domains may confer bindingspecificity, regulating the activity of various caspases, for example.

Nuclear factor-κB (NF-κB) is a transcription factor that is expressed inmany cell types and activates genes that have NF-κB sites in theirpromoters. Molecules that regulate NF-κB activation play a critical rolein both apoptosis and in the stress-response of cells. With respect tostress-reponse, NF-κB activates genes that control immune defensemechanisms and inflammation. The CARD-containing proteins RICK, CARD-4and Bcl-10 also induce activation of the NF-κB transcription factorsuggesting that CARD/CARD signaling complexes regulate activation of theIKK complex (Inohara et al. 1998 Proc. Natl. Acad Sci. USA 273:12296;Bertin et al. 1999 J. Biol. Chem. 224:12955; Willis et al. 1999 Cell 96:33). In unstimulated cells, NF-?B is found sequestered in the cytoplasmthrough interactions with inhibitory IκB proteins. Inhibition isrelieved by the phosphorylation and proteosomal degradation of IκBproteins by proinflammatory cytokines. Phosphorylation is mediated bythe IKK complex which is comprised of at least three major proteins: twokinases designated IKKα and IKKβ that directly phosphorylate the IκBinhibitory proteins, and a noncatalytic subunit called IKKγ thatfunctions to link the IKKs to upstream regulatory molecules (Zhang etal., 2000). Recently, RICK has been found to function as upstreamregulatory molecules of the IKK complex (Inohara et al. 2000 J. Biol.Chem. 275:27823). RICK interacts directly with IKKγ suggesting that itfunctions as signaling adaptor between the IKK complex and an upstreamCARD-containing NF-κB activator. Indeed, CARD-4 forms a CARD/CARDsignaling complex with RICK that induces activation of the IKK complexand the subsequent release of NF-κB (Bertin et al. 1999 J. Biol. Chem.224:12955; Inohara et al. 1999 J. Biol. Chem. 274:14566; Inohara et al.2000 J. Biol. Chem. 275:27823).

At least two dozen stimuli that activate NF-?B are known, includingcytokines, protein kinase C activators, oxidants, viruses, and immunesystem stimuli. NK-?B is stimulated via signaling through the tumornecrosis factor family receptors (TNFRs) and the interleukin-1/Tollreceptor. Tumor necrosis factor family members bind to their cognatereceptors, including Fas (CD95/APO-1), TRAMP (DR3/WSL-1/AIR/LARD), CD37,CD30, CD40, TNFR1 and TNFR2, and regulate apoptosis, cell proliferation,and proinflammatory responses. For example, the proinflammatorycytokines TNF-α and IL-1 induce NF-κB activation by binding theircell-surface receptors and activating the NF-κB-inducing kinase, NIK. Inthe case of TNF-α, binding to TNF-R1 induces aggregation of its deathdomain and assembly of a signaling complex containing TRADD, TRAF2, andRIP. Binding of IL-1 to its receptor, IL-1R, induces aggregation of thereceptor and assembly of a signaling complex which includes AcP, MyD88,IRAK1, IRAK2, and TRAF6. Both the TNF-R1 complex and the IL-1R complextrigger activation of NIK. Activated NIK phosphorylates the IkB kinasesIkB-a and IkB-b which phosphorylate IkB, leading to its degradation and,as a consequence, the activation of NF-κB.

Fas, a cell surface receptor that is a member of the TNFR family, caninduce apoptosis upon binding with its ligand, FasL (CD95L). Fasinteracts with FADD (MORT) via death domains present in both proteins.When bound to Fas, FADD interacts with caspase-8 (FLICE/MACH/Mch5)through death effector domains present in both proteins. The complex ofFas, FADD and caspase-8 is referred to as the death-inducing signalingcomplex (DISC). Recently, FLASH, a protein having a DRD as well as aCED-4-like domain, has been identified as a component of DISC that isrequired for caspase-8 activation during Fas-mediated apoptosis (Imai etal. (1999) Nature 398:777-85). In the DISC, caspase-8 undergoesoligomerization-dependent autoproteolysis, leading to activation.Activated caspase-8 cleaves several effector caspases, includingcaspase-3, caspase-6, and caspase-7, by proteolytic cleavage. Theseeffector caspases cleave various death substrates involved in themorphological changes and DNA fragmentation that is central toapoptosis.

Transient expression of FLASH activates caspase-8. However, a truncatedform of FLASH lacking either its DRD or CED-4-like domain does not allowactivation of caspase-8 or Fas-mediated apoptosis. Thus, it appears thatFLASH is involved in both Fas- and TNF-induced apoptosis mediated byactivated caspase-8 (Imai et al. (1999) Nature 398:777-85).

Bcl-10 (mE10/CIPER/CLAP/c-CARMEN) is a CARD domain containingpro-apoptotic protein that induces NF-κB activation (Koseki et al.(1999) J. Biol. Chem. 274:9955-61; Yan et al. (1999) J. Biol. Chem.274:10287-92; Thome et al. (1999) J. Biol. Chem. 274:9962-68;Srinivasula et al. (1999) J. Biol. Chem. 274:17946-54)). Bcl-10activates NF-κB by acting upstream of NIK and IkB kinase (Srinivasula etal., supra). Significantly, Bcl-10 is involved in t(1;14)(p22;q23) ofMALT B cell lymphoma (Willis et al. (1999) Cell 96:35-45; Zhang et al.(1999) Nat. Genet. 22:63-8). Bcl-10 expressed in MALT lymphoma exhibitsa frameshift mutation that causes truncation of Bcl-10 distal to itsCARD domain. The truncated form of Bcl-10 activates NF-κB, but does notinduce apoptosis (Willis et al. (1999) Cell 96:35-45). Expression ofNF-κB is associated with suppression of apoptosis and increased cellsurvival in certain systems. Thus, mutant Bcl-10 may promote continuedcell proliferation by two different mechanisms. Bcl-10 mutations similarto that observed in MALT lymphoma occur in many other tumor types,suggesting that Bcl-10 may be commonly involved in malignancy. Bcl-10has a bipartite structure consisting of an N-terminal CARD domain and aC-terminal effector domain that mediates activation of NF-κB.

Bcl-10 has been implicated as a positive regulator of lymphocyteactivation and proliferation triggered by antigen receptor engagement(Ruland et al. (2001) Cell 104:33-42). Mice lacking Bcl-10 are severelyimmunodeficient, e.g., impaired humoral and cellular immune responses,and have lymphocytes defective in antigen induced NF-kB activation.Thus, Bcl-10 appears to act as a mediator of NF-kB activation inresponse to antigen receptor signaling in B and T cells. In addition,approximately one third of Bcl-10 deficient embryos developedexencephaly, implicating a role for Bcl-10 in normal CNS development,possibly via positive regulation of neuronal survival (Ruland et al.supra).

Murine FLASH is a protein involved in Fas-mediated activation ofcaspase-8 during apoptosis (Imani et al. (1999) Nature 398:777-85).Transient expression of murine FLASH activates caspase-8. It appearsthat the DRD domain (amino acids 1584-1751) and the CED-4-like domain(amino acids 939-1191) of murine FLASH are required for activation ofcaspase-8. In addition, the exencephaly seen in about one third ofbcl-10−/− embryos suggests that Bcl-10 may be involded in the positiveregulation of neuronal survival.

NF-kB and the NF-kB pathway have been implicated in mediating chronicinflammation in inflammatory diseases such as asthma, ulcerativecolitis, rheumatoid arthritis (Barnes & Epstein (1997) New EnglandJournal of Medicine 336:1066) and inhibiting NF-kB or NF-kB pathways maybe an effective way of treating these diseases. Binding sites for thetranscription factor NF-kB are present in the promoter regions of thegenes of many of the proinflammatory cytokines, chemokines, enzymes,immune receptors, and adhesion molecules important in inducing acuteinflammatory responses associated with critical illnesses. Becauseincreased activation of NF-kB can lead to enhanced expression ofproinflammatory mediators, NF-kB activation may be an important event inthe development of, for example, multiple organ dysfunction associatedwith infection, blood loss, and ischemia-reperfusion injury (Abraham(2000) Crit Care Med 28(4 Suppl):N100-4).

NF-kB and the NF-kB pathway have also been implicated in atherosclerosis(Navab et al. (1995) American Journal of Cardiology 76:18C), especiallyin mediating fatty streak formation, and inhibiting NF-kB or NF-kBpathways may be an effective therapy for atherosclerosis. Among thegenes activated by NF-kB are cIAP-1, cIAP-2, TRAF1, and TRAF2, all ofwhich have been shown to protect cells from TNF-I induced cell death(Wang et al. (1998) Science 281:1680-83).

SUMMARY OF THE INVENTION

The invention features nucleic acid molecules encoding rat CARD-9, humanCARD-9, human CARD-10, and human CARD-11. These proteins, like manyothers having a CARD domain, play roles in apoptotic and inflammatorysignaling pathways. CARD-9, CARD-10, and CARD-11 participate in thenetwork of interactions that modulate caspase activity. Upon activation,CARD-9, CARD-10, and CARD-11 likely bind and activate a CARD containingprotein via a CARD-CARD interaction leading to a modulation of apoptosisand or stress related pathways (e.g., NF-κB activation).

CARD-9, CARD-10, and CARD 11 molecules are useful as modulating agentsin regulating a variety of cellular processes including cell growth andcell death. In one aspect, this invention provides isolated nucleic acidmolecules encoding CARD-9, CARD10, or CARD-11 proteins or biologicallyactive portions thereof, as well as nucleic acid fragments suitable asprimers or hybridization probes for the detection of CARD-9, CARD10, orCARD-11 encoding nucleic acids.

The invention encompasses methods of diagnosing and treating patientswho are suffering from a disorder associated with an abnormal level orrate (undesirably high or undesirably low) of apoptotic cell death,abnormal activity of the Fas/APO-1 receptor complex, abnormal activityof the TNF receptor complex, or abnormal activity of a caspase byadministering a compound that modulates the expression of CARD-9,CARD-10, or CARD-11 (at the DNA, mRNA or protein level, e.g., byaltering mRNA splicing) or by altering the activity of CARD-9, CARD-10,or CARD-11. Examples of such compounds include small molecules,antisense nucleic acid molecules, ribozymes, and polypeptides.

Certain disorders are associated with an increased number of survivingcells, which are produced and continue to survive or proliferate whenapoptosis is inhibited or occurs at an undesirably low rate. CARD-9,CARD-10, or CARD-11 and compounds that modulate the expression oractivity of CARD-9, CARD-10, or CARD-11 can be used to treat or diagnosesuch disorders. These disorders include cancer (particularly follicularlymphomas, chronic myelogenous leukemia, melanoma, colon cancer, lungcarcinoma, carcinomas associated with mutations in p53, andhormone-dependent tumors such as breast cancer, prostate cancer, andovarian cancer). Such compounds can also be used to treat viralinfections (such as those caused by herpesviruses, poxviruses, andadenoviruses). Failure to remove autoimmune cells that arise duringdevelopment or that develop as a result of somatic mutation during animmune response can result in autoimmune disease. Thus, autoimmunedisorders can be caused by an undesirably low levels of apoptosis.Accordingly, CARD-9, CARD10, or CARD-11 and modulators of CARD-9,CARD-10, or CARD-11 activity or expression can be used to treatautoimmune disorders (e.g., systemic lupus erythematosis,immune-mediated glomerulonephritis, and arthritis).

Many diseases are associated with an undesirably high rate of apoptosis.CARD-9, CARD10, or CARD-11 and modulators of CARD-9, CARD-10, or CARD-11expression or activity can be used to treat or diagnose such disorders.For example, populations of cells are often depleted in the event ofviral infection, with perhaps the most dramatic example being the celldepletion caused by the human immunodeficiency virus (HIV).Surprisingly, most T cells that die during HIV infections do not appearto be infected with HIV. Although a number of explanations have beenproposed, recent evidence suggests that stimulation of the CD4 receptorresults in the enhanced susceptibility of uninfected T cells to undergoapoptosis. A wide variety of neurological diseases are characterized bythe gradual loss of specific sets of neurons. Such disorders includeAlzheimer's disease, Parkinson's disease, amyotrophic lateral sclerosis(ALS) retinitis pigmentosa, spinal muscular atrophy, and various formsof cerebellar degeneration. The cell loss in these diseases does notinduce an inflammatory response, and apoptosis appears to be themechanism of cell death. In addition, a number of hematologic diseasesare associated with a decreased production of blood cells. Thesedisorders include anemia associated with chronic disease, aplasticanemia, chronic neutropenia, and the myelodysplastic syndromes.Disorders of blood cell production, such as myelodysplastic syndrome andsome forms of aplastic anemia, are associated with increased apoptoticcell death within the bone marrow. These disorders could result from theactivation of genes that promote apoptosis, acquired deficiencies instromal cells or hematopoietic survival factors, or the direct effectsof toxins and mediators of immune responses. Two common disordersassociated with cell death are myocardial infarctions and stroke. Inboth disorders, cells within the central area of ischemia, which isproduced in the event of acute loss of blood flow, appear to die rapidlyas a result of necrosis. However, outside the central ischemic zone,cells die over a more protracted time period and morphologically appearto die by apoptosis.

Proteins containing a CARD domain are thought to be involved in variousinflammatory disorders. Accordingly, CARD-9, CARD-10, or CARD-11polypeptides, nucleic acids and modulators of CARD-9, CARD-10, orCARD-11 expression or activity can be used to treat immune disorders.Such immune disorders include, but are not limited to, chronicinflammatory diseases and disorders, such as Crohn's disease, reactivearthritis, including Lyme disease, insulin-dependent diabetes,organ-specific autoimmunity, including multiple sclerosis, Hashimoto'sthyroiditis and Grave's disease, contact dermatitis, psoriasis, graftrejection, graft versus host disease, sarcoidosis, atopic conditions,such as asthma and allergy, including allergic rhinitis,gastrointestinal allergies, including food allergies, eosinophilia,conjunctivitis, glomerular nephritis, certain pathogen susceptibilitiessuch as helminthic (e.g., leishmaniasis), certain viral infections,including HIV, and bacterial infections, including tuberculosis andlepromatous leprosy.

In addition to the aforementioned disorders, CARD-9, CARD-10, or CARD-11polypeptides, nucleic acids, and modulators of CARD-9, CARD-10, orCARD-11 expression or activity can be used to treat disorders of cellsignaling and disorders of tissues in which CARD-9, CARD-10, or CARD-11is expressed.

The invention features a nucleic acid molecule which is at least 45% (or55%, 65%, 75%, 85%, 95%, or 98%) identical to the nucleotide sequenceshown in SEQ ID NO: 1, SEQ ID NO: 3, SEQ ID NO: 4, SEQ ID NO: 6, SEQ IDNO: 7, SEQ ID NO: 9, SEQ ID NO: 10, SEQ ID NO: 12, or a complementthereof.

The invention features a nucleic acid molecule which includes a fragmentof at least 150 (300, 325, 350, 375, 400, 425, 450, 500, 550, 600, 650,700, 800, 900, 1000, 1200, 1400, 1600, 1800, 2000, 2200, 2400, 2600,2800, 3000, 3200, 3400, 3600, 3800, 4000, 4200, or 4250) nucleotides ofthe nucleotide sequence shown in SEQ ID NO: 1, SEQ ID NO: 3, SEQ ID NO:4, SEQ ID NO: 6, SEQ ID NO: 7, SEQ ID NO: 9, SEQ ID NO: 10, SEQ ID NO:12, or a complement thereof.

In an embodiment, a CARD-9 nucleic acid molecule has the nucleotidesequence shown in SEQ ID NO: 1, or SEQ ID NO: 3.

Also within the invention is a nucleic acid molecule which encodes afragment of a polypeptide having the amino acid sequence of SEQ ID NO:2.

The invention includes a nucleic acid molecule which encodes a naturallyoccurring allelic variant of a polypeptide comprising the amino acidsequence of SEQ ID NO: 2, wherein the nucleic acid molecule hybridizesto a nucleic acid molecule consisting of SEQ ID NO: 1, or SEQ ID NO: 3.

In general, an allelic variant of a gene will be readily identifiable asmapping to the same chromosomal location as said gene.

The invention also includes a nucleic acid molecule encoding a naturallyoccurring polypeptide, wherein the nucleic acid hybridizes to a nucleicacid molecule consisting of SEQ ID NO: 3 under stringent conditions(e.g., hybridization in 6×sodium chloride/sodium citrate (SSC) at about60° C., followed by one or more washes in 0.2×SSC, 0.1% SDS at 65° C.),and wherein the nucleic acid encodes a polypeptide of 533-539 aminoacids in length, preferably 536 amino acids, having a molecular weightof approximately 62.2 kD prior to post-translational modifications.Thus, the invention encompasses a nucleic acid molecule which includesthe sequence of the protein coding region of a naturally occurring mRNA(or the corresponding cDNA sequence) that is expressed in a human cell.

Also within the invention are: an isolated CARD-9 protein having anamino acid sequence that is at least about 65%, preferably 75%, 85%,95%, or 98% identical to the amino acid sequence of SEQ ID NO: 2; anisolated CARD-9 protein having an amino acid sequence that is at leastabout 65%, preferably 75%, 85%, 95%, or 98% identical to the CARD domainof SEQ ID NO: 2 (e.g., about amino acid residues 7-98 of SEQ ID NO: 2);an isolated CARD-9 protein having an amino acid sequence that is atleast about 65%, preferably 75%, 85%, 95%, or 98% identical to thecoiled-coil domain of SEQ ID NO: 2 (e.g., about amino acid residues140-416 of SEQ ID NO: 2); an isolated CARD-9 protein having an aminoacid sequence that is at least about 65%, preferably 75%, 85%, 95%, or98% identical to the indole-3-glycerol phosphate synthase homologydomain of SEQ ID NO: 2 (e.g., about amino acid residues 197-213 of SEQID NO: 2); and an isolated CARD-9 protein having an amino acid sequencethat is at least about 65%, preferably 75%, 85%, 95%, or 98% identicalto the cysteine rich repeat homology domain of SEQ ID NO: 2 (e.g., aboutamino acid residues 285-338 of SEQ ID NO: 2).

In an embodiment, a CARD-9 nucleic acid molecule has the nucleotidesequence shown in SEQ ID NO: 4, or SEQ ID NO: 6.

Also within the invention is a nucleic acid molecule which encodes afragment of a polypeptide having the amino acid sequence of SEQ ID NO:5.

The invention includes a nucleic acid molecule which encodes a naturallyoccurring allelic variant of a polypeptide comprising the amino acidsequence of SEQ ID NO: 5, wherein the nucleic acid molecule hybridizesto a nucleic acid molecule consisting of SEQ ID NO: 4, or SEQ ID NO: 6.

In general, an allelic variant of a gene will be readily identifiable asmapping to the same chromosomal location as said gene.

The invention also includes a nucleic acid molecule encoding a naturallyoccurring polypeptide, wherein the nucleic acid hybridizes to a nucleicacid molecule consisting of SEQ ID NO: 6 under stringent conditions(e.g., hybridization in 6×sodium chloride/sodium citrate (SSC) at about60° C., followed by one or more washes in 0.2×SSC, 0.1% SDS at 65° C.),and wherein the nucleic acid encodes a polypeptide of 533-539 aminoacids in length, preferably 536 amino acids, having a molecular weightof approximately 62.2 kD prior to post-translational modifications.Thus, the invention encompasses a nucleic acid molecule which includesthe sequence of the protein coding region of a naturally occurring mRNA(or the corresponding cDNA sequence) that is expressed in a human cell.

Also within the invention are: an isolated CARD-9 protein having anamino acid sequence that is at least about 65%, preferably 75%, 85%,95%, or 98% identical to the amino acid sequence of SEQ ID NO: 5; anisolated CARD-9 protein having an amino acid sequence that is at leastabout 65%, preferably 75%, 85%, 95%, or 98% identical to the CARD domainof SEQ ID NO: 5 (e.g., about amino acid residues 7-98 of SEQ ID NO: 5);an isolated CARD-9 protein having an amino acid sequence that is atleast about 65%, preferably 75%, 85%, 95%, or 98% identical to thecoiled-coil domain of SEQ ID NO: 5 (e.g., about amino acid residues140-416 of SEQ ID NO: 5); an isolated CARD-9 protein having an aminoacid sequence that is at least about 65%, preferably 75%, 85%, 95%, or98% identical to the indole-3-glycerol phosphate synthase homologydomain of SEQ ID NO: 5 (e.g., about amino acid residues 197-213 of SEQID NO: 5); and an isolated CARD-9 protein having an amino acid sequencethat is at least about 65%, preferably 75%, 85%, 95%, or 98% identicalto the cysteine rich repeat homology domain of SEQ ID NO: 5 (e.g., aboutamino acid residues 285-338 of SEQ ID NO: 5).

In an embodiment, a CARD-10 nucleic acid molecule has the nucleotidesequence shown in SEQ ID NO: 7, or SEQ ID NO: 9.

Also within the invention is a nucleic acid molecule which encodes afragment of a polypeptide having the amino acid sequence of SEQ ID NO:8.

The invention includes a nucleic acid molecule which encodes a naturallyoccurring allelic variant of a polypeptide comprising the amino acidsequence of SEQ ID NO: 8, wherein the nucleic acid molecule hybridizesto a nucleic acid molecule consisting of SEQ ID NO: 7, or SEQ ID NO: 9.

In general, an allelic variant of a gene will be readily identifiable asmapping to the same chromosomal location as said gene.

The invention also includes a nucleic acid molecule encoding a naturallyoccurring polypeptide, wherein the nucleic acid hybridizes to a nucleicacid molecule consisting of SEQ ID NO: 9 under stringent conditions(e.g., hybridization in 6×sodium chloride/sodium citrate (SSC) at about60° C., followed by one or more washes in 0.2×SSC, 0.1% SDS at 65° C.),and wherein the nucleic acid encodes a polypeptide of 1029-1035 aminoacids in length, preferably 1032 amino acids, having a molecular weightof approximately 115.9 kD prior to post-translational modifications.Thus, the invention encompasses a nucleic acid molecule which includesthe sequence of the protein coding region of a naturally occurring mRNA(or the corresponding cDNA sequence) that is expressed in a human cell.

Also within the invention are: an isolated CARD-10 protein having anamino acid sequence that is at least about 65%, preferably 75%, 85%,95%, or 98% identical to the amino acid sequence of SEQ ID NO: 8; anisolated CARD-10 protein having an amino acid sequence that is at leastabout 85%, 95%, or 98% identical to the CARD domain of SEQ ID NO: 8(e.g., about amino acid residues 23-123 of SEQ ID NO: 8); an isolatedCARD-10 protein having an amino acid sequence that is at least about85%, 95%, or 98% identical to the coiled-coil domain of SEQ ID NO: 8(e.g., about amino acid residues 147-457 of SEQ ID NO: 8); an isolatedCARD-10 protein having an amino acid sequence that is at least about85%, 95%, or 98% identical to the SH3 domain of SEQ ID NO: 8 (e.g.,about amino acid residues 704-772 of SEQ ID NO: 8); an isolated CARD-10protein having an amino acid sequence that is at least about 85%, 95%,or 98% identical to the guanylate kinase (GUK) domain of SEQ ID NO: 8(e.g., about amino acid residues 830-1032 of SEQ ID NO: 8); and anisolated CARD-10 protein having an amino acid sequence that is at leastabout 85%, 95%, or 98% identical to the tropomyosin domain of SEQ ID NO:8 (e.g., about amino acid residues 366-398 of SEQ ID NO: 8);

In an embodiment, a CARD-11 nucleic acid molecule has the nucleotidesequence shown in SEQ ID NO: 10, or SEQ ID NO: 12.

Also within the invention is a nucleic acid molecule which encodes afragment of a polypeptide having the amino acid sequence of SEQ ID NO:11.

The invention includes a nucleic acid molecule which encodes a naturallyoccurring allelic variant of a polypeptide comprising the amino acidsequence of SEQ ID NO: 11, wherein the nucleic acid molecule hybridizesto a nucleic acid molecule consisting of SEQ ID NO: 10 or SEQ ID NO: 12.

In general, an allelic variant of a gene will be readily identifiable asmapping to the same chromosomal location as said gene.

The invention also includes a nucleic acid molecule encoding a naturallyoccurring polypeptide, wherein the nucleic acid hybridizes to a nucleicacid molecule consisting of SEQ ID NO: 12 under stringent conditions(e.g., hybridization in 6×sodium chloride/sodium citrate (SSC) at about60° C., followed by one or more washes in 0.2×SSC, 0.1% SDS at 65° C.),and wherein the nucleic acid encodes a polypeptide of 1144-1150 aminoacids in length, preferably 1147 amino acids, having a molecular weightof approximately 132.6 kD prior to post-translational modifications.Thus, the invention encompasses a nucleic acid molecule which includesthe sequence of the protein coding region of a naturally occurring mRNA(or the corresponding cDNA sequence) that is expressed in a human cell.

Also within the invention are: an isolated CARD-11 protein having anamino acid sequence that is at least about 65%, preferably 75%, 85%,95%, or 98% identical to the amino acid sequence of SEQ ID NO: 11; anisolated CARD-11 protein having an amino acid sequence that is at leastabout 65%, preferably 75%, 85%, 95%, or 98% identical to the CARD domainof SEQ ID NO: 11 (e.g., about amino acid residues 6-112 of SEQ ID NO:11); an isolated CARD-11 protein having an amino acid sequence that isat least about 65%, preferably 75%, 85%, 95%, or 98% identical to thecoiled-coil domain of SEQ ID NO: 11 (e.g., about amino acid residues130-431 of SEQ ID NO: 11); an isolated CARD-11 protein having an aminoacid sequence that is at least about 65%, preferably 75%, 85%, 95%, or98% identical to the PDZ domain of SEQ ID NO: 11 (e.g., about amino acidresidues 635-748 of SEQ ID NO: 11); an isolated CARD-11 protein havingan amino acid sequence that is at least about 65%, preferably 75%, 85%,95%, or 98% identical to the SH3 domain of SEQ ID NO: 11 (e.g., aboutamino acid residues 766-834 of SEQ ID NO: 11); and an isolated CARD-11protein having an amino acid sequence that is at least about 65%,preferably 75%, 85%, 95%, or 98% identical to the guanylate kinase (GUK)domain of SEQ ID NO: 11 (e.g., about amino acid residues 882-1147 of SEQID NO: 11);

Also within the invention are: an isolated CARD-9 protein which isencoded by a nucleic acid molecule having a nucleotide sequence that isat least about 65%, preferably 75%, 85%, or 95% identical to SEQ ID NO:3; an isolated CARD-9 protein which is encoded by a nucleic acidmolecule having a nucleotide sequence at least about 65% preferably 75%,85%, or 95% identical to the CARD domain encoding portion of SEQ ID NO:3; an isolated CARD-9 protein which is encoded by a nucleic acidmolecule having a nucleotide sequence at least about 65% preferably 75%,85%, or 95% identical to the coiled-coil domain encoding portion of SEQID NO: 3; an isolated CARD-9 protein which is encoded by a nucleic acidmolecule having a nucleotide sequence at least about 65% preferably 75%,85%, or 95% identical to the indole-3-glycerol phosphate synthasehomology domain encoding portion of SEQ ID NO: 3; an isolated CARD-9protein which is encoded by a nucleic acid molecule having a nucleotidesequence at least about 65% preferably 75%, 85%, or 95% identical to thecysteine rich repeat homology domain encoding portion of SEQ ID NO: 3;and an isolated CARD-9 protein which is encoded by a nucleic acidmolecule having a nucleotide sequence which hybridizes under stringenthybridization conditions to a nucleic acid molecule having thenucleotide sequence of SEQ ID NO: 3.

Also within the invention are: an isolated CARD-9 protein which isencoded by a nucleic acid molecule having a nucleotide sequence that isat least about 65%, preferably 75%, 85%, or 95% identical to SEQ ID NO:6; an isolated CARD-9 protein which is encoded by a nucleic acidmolecule having a nucleotide sequence at least about 65% preferably 75%,85%, or 95% identical to the CARD domain encoding portion of SEQ ID NO:6; an isolated CARD-9 protein which is encoded by a nucleic acidmolecule having a nucleotide sequence at least about 65% preferably 75%,85%, or 95% identical to the coiled-coil domain encoding portion of SEQID NO: 6; an isolated CARD-9 protein which is encoded by a nucleic acidmolecule having a nucleotide sequence at least about 65% preferably 75%,85%, or 95% identical to the indole-3-glycerol phosphate synthasehomology domain encoding portion of SEQ ID NO: 6; an isolated CARD-9protein which is encoded by a nucleic acid molecule having a nucleotidesequence at least about 65% preferably 75%, 85%, or 95% identical to thecysteine rich repeat homology domain encoding portion of SEQ ID NO: 6;and an isolated CARD-9 protein which is encoded by a nucleic acidmolecule having a nucleotide sequence which hybridizes under stringenthybridization conditions to a nucleic acid molecule having thenucleotide sequence of SEQ ID NO: 6.

Also within the invention are: an isolated CARD-10 protein which isencoded by a nucleic acid molecule having a nucleotide sequence that isat least about 65%, preferably 75%, 85%, or 95% identical to SEQ ID NO:9; an isolated CARD-10 protein which is encoded by a nucleic acidmolecule having a nucleotide sequence at least about 65% preferably 75%,85%, or 95% identical to the CARD domain encoding portion of SEQ ID NO:9; an isolated CARD-10 protein which is encoded by a nucleic acidmolecule having a nucleotide sequence at least about 65% preferably 75%,85%, or 95% identical to the coiled-coil domain encoding portion of SEQID NO: 9; an isolated CARD-10 protein which is encoded by a nucleic acidmolecule having a nucleotide sequence at least about 65% preferably 75%,85%, or 95% identical to the SH3 domain encoding portion of SEQ ID NO:9; an isolated CARD-10 protein which is encoded by a nucleic acidmolecule having a nucleotide sequence at least about 65% preferably 75%,85%, or 95% identical to the guanylate kinase (GUK) domain encodingportion of SEQ ID NO: 9; an isolated CARD-10 protein which is encoded bya nucleic acid molecule having a nucleotide sequence at least about 65%preferably 75%, 85%, or 95% identical to the tropomyosin domain encodingportion of SEQ ID NO: 9; and an isolated CARD-10 protein which isencoded by a nucleic acid molecule having a nucleotide sequence whichhybridizes under stringent hybridization conditions to a nucleic acidmolecule having the nucleotide sequence of SEQ ID NO: 9.

Also within the invention are: an isolated CARD-11 protein which isencoded by a nucleic acid molecule having a nucleotide sequence that isat least about 65%, preferably 75%, 85%, or 95% identical to SEQ ID NO:12; an isolated CARD-11 protein which is encoded by a nucleic acidmolecule having a nucleotide sequence at least about 65% preferably 75%,85%, or 95% identical to the CARD domain encoding portion of SEQ ID NO:12; an isolated CARD-11 protein which is encoded by a nucleic acidmolecule having a nucleotide sequence at least about 65% preferably 75%,85%, or 95% identical to the coiled-coil domain encoding portion of SEQID NO: 12; an isolated CARD-11 protein which is encoded by a nucleicacid molecule having a nucleotide sequence at least about 65% preferably75%, 85%, or 95% identical to the PDZ domain encoding portion of SEQ IDNO: 12; an isolated CARD-11 protein which is encoded by a nucleic acidmolecule having a nucleotide sequence at least about 65% preferably 75%,85%, or 95% identical to the SH3 domain encoding portion of SEQ ID NO:12; an isolated CARD-11 protein which is encoded by a nucleic acidmolecule having a nucleotide sequence at least about 65% preferably 75%,85%, or 95% identical to the guanylate kinase (GUK) domain encodingportion of SEQ ID NO: 12; and an isolated CARD-11 protein which isencoded by a nucleic acid molecule having a nucleotide sequence whichhybridizes under stringent hybridization conditions to a nucleic acidmolecule having the nucleotide sequence of SEQ ID NO: 12.

The CARD-9, CARD-10, or CARD-11 polypeptides, nucleic acids, andantibodies of the invention may be useful for mapping the location ofthe CARD-9, CARD-10, or CARD-11 genes as well as the location of genesassociated with diseases that map to the same region as the CARD-9,CARD-10, or CARD-11 genes. The CARD-10 gene is located in chromosome22q13.1. The CARD-10 polypeptides, nucleic acids, and antibodies of theinvention may be useful for mapping the location of the CARD-10 gene aswell as the location of genes associated with the following diseases:spinocerebellar ataxia 10 and meningioma 1, both of which map tochromosome 22 in the region of the CARD-10 gene.

Another embodiment of the invention features CARD-9, CARD-10, or CARD-11nucleic acid molecules which specifically detect CARD-9, CARD-10, orCARD-11 nucleic acid molecules, relative to nucleic acid moleculesencoding other members of the CARD superfamily. For example, in oneembodiment, a CARD-9, CARD-10, or CARD-11 nucleic acid moleculehybridizes under stringent conditions to a nucleic acid moleculecomprising the nucleotide sequence of SEQ ID NO: 1, SEQ ID NO: 3, SEQ IDNO: 4, SEQ ID NO: 6, SEQ ID NO: 7, SEQ ID NO: 9, SEQ ID NO: 10, SEQ IDNO: 12, or a complement thereof. In another embodiment, the CARD-9,CARD-10, or CARD-11 nucleic acid molecule is at least 300 (350, 400,450, 500, 550, 600, 650, 700, 800, 900, 1000, 1200, 1400, 1600, 1800,2000, 2200, 2400, 2600, 2800, 3000, 3200, 3400, 3600, 3800, 4000, 4200,or 4250) nucleotides in length and hybridizes under stringent conditionsto a nucleic acid molecule comprising the nucleotide sequence shown inSEQ ID NO: 1, SEQ ID NO: 3, SEQ ID NO: 4, SEQ ID NO: 6, SEQ ID NO: 7,SEQ ID NO: 9, SEQ ID NO: 10, SEQ ID NO: 12, or a complement thereof. Inanother embodiment, an isolated CARD-9, CARD-10, or CARD-11 nucleic acidmolecule comprises the CARD domain encoding portion of SEQ ID NO: 3, SEQID NO: 6, SEQ ID NO: 9, SEQ ID NO: 12 or a complement thereof. In yetanother embodiment, the invention provides an isolated nucleic acidmolecule which is antisense to the coding strand of a CARD-9, CARD-10,or CARD-11 nucleic acid.

Another aspect of the invention provides a vector, e.g., a recombinantexpression vector, comprising a CARD-9, CARD-10, or CARD-11 nucleic acidmolecule of the invention. In another embodiment the invention providesa host cell containing such a vector. The invention also provides amethod for producing CARD-9, CARD-10, or CARD-11 protein by culturing,in a suitable medium, a host cell of the invention containing arecombinant expression vector such that a CARD-9, CARD-10, or CARD-11protein is produced.

Another aspect of this invention features isolated or recombinantCARD-9, CARD-10, or CARD-11 proteins and polypeptides. Preferred CARD-9,CARD-10, or CARD-11 proteins and polypeptides possess at least onebiological activity possessed by naturally occurring human CARD-9,CARD-10, or CARD-11, e.g., (1) the ability to form protein:proteininteractions with proteins in the apoptotic signaling pathway; (2) theability to form CARD-CARD interactions with proteins in the apoptoticsignaling pathway, e.g., Bcl-10; (3) the ability to bind a CARD-9,CARD-10, or CARD-11 ligand; (4) the ability to bind to an intracellulartarget; and (5) the ability to activate the NF-κB pathway. Otheractivities include: (1) modulation of cellular proliferation; (2)modulation of cellular differentiation; (3) modulation of cellulardeath; (4) modulation of the NF-κB pathway; and (5) modulation ofstress-responsive signaling pathways.

The CARD-9, CARD-10, or CARD-11 proteins of the present invention, orbiologically active portions thereof, can be operatively linked to anon-CARD-9, CARD-10, or CARD-11 polypeptide (e.g., heterologous aminoacid sequences) to form CARD-9, CARD-10, or CARD-11 fusion proteins,respectively. The invention further features antibodies thatspecifically bind CARD-9, CARD-10, or CARD-11 proteins, such asmonoclonal or polyclonal antibodies. In addition, the CARD-9, CARD-10,or CARD-11 proteins or biologically active portions thereof can beincorporated into pharmaceutical compositions, which optionally includepharmaceutically acceptable carriers.

In another aspect, the present invention provides a method for detectingthe presence of CARD-9, CARD-10, or CARD-11 activity or expression in abiological sample by contacting the biological sample with an agentcapable of detecting an indicator of CARD-9, CARD-10, or CARD-11activity such that the presence of CARD-9, CARD-10, or CARD-11 activityis detected in the biological sample.

In another aspect, the invention provides a method for modulatingCARD-9, CARD-10, or CARD-11 activity comprising contacting a cell withan agent that modulates (inhibits or stimulates) CARD-9, CARD-10, orCARD-11 activity or expression such that CARD-9, CARD-10, or CARD-11activity or expression in the cell is modulated. Examples of CARD-9,CARD-10, or CARD-11 activities include the ability to bind to Bcl-10,stimulate the phosphorylation of Bcl-10, and stimulate the activation ofNF-kB. In one embodiment, the agent is an antibody that specificallybinds to CARD-9, CARD-10, or CARD-11 protein. In another embodiment, theagent modulates (increases or decreases) expression of CARD-9, CARD-10,or CARD-11 by modulating transcription of a CARD-9, CARD-10, or CARD-11gene, splicing of a CARD-9, CARD-10, or CARD-11 mRNA, or translation ofa CARD-9, CARD-10, or CARD-11 mRNA. In yet another embodiment, the agentis a nucleic acid molecule having a nucleotide sequence that isantisense to the coding strand of the CARD-9, CARD10, or CARD-11 mRNA orthe CARD-9, CARD-10, or CARD-11 gene.

In one embodiment, the methods of the present invention are used totreat a subject having a disorder characterized by aberrant CARD-9,CARD-10, or CARD-11 protein or nucleic acid expression or activity orrelated to CARD-9, CARD-10, or CARD-11 expression or activity byadministering an agent which is a CARD-9, CARD-10, or CARD-11 modulatorto the subject. In one embodiment, the CARD-9, CARD-10, or CARD-11modulator is a CARD-9, CARD-10, or CARD-11 protein. In anotherembodiment the CARD-9, CARD-10, or CARD-11 modulator is a CARD-9,CARD-10, or CARD-11 nucleic acid molecule. In other embodiments, theCARD-9, CARD-10, or CARD-11 modulator is a peptide, peptidomimetic, orother small molecule.

The present invention also provides a diagnostic assay for identifyingthe presence or absence of a genetic lesion or mutation characterized byat least one of: (i) aberrant modification or mutation of a geneencoding a CARD-9, CARD-10, or CARD-11 protein; (ii) mis-regulation of agene encoding a CARD-9, CARD-10, or CARD-11 protein; (iii) aberrant RNAsplicing; and (iv) aberrant post-translational modification of a CARD-9,CARD-10, or CARD-11 protein, wherein a wild-type form of the geneencodes a protein with a CARD-9, CARD-10, or CARD-11 activity.

In another aspect, the invention provides a method for identifying acompound that modulates (increases or decreases) the ability of CARD-11to stimulate the phosphorylation of Bcl-10. In general, the methodentails measuring the ability of CARD-11 to stimulate thephosphorylation of Bcl-10 in the presence and absence of a test compoundor test compounds, and identifying the compound or compounds thatmodulate the ability of CARD-11 to stimulate the phosphorylation ofBcl-10.

In another aspect, the invention provides a method for identifying acompound that binds to or modulates the activity of a CARD-9, CARD-10,or CARD-11 protein. In general, such methods entail measuring abiological activity of a CARD-9, CARD-10, or CARD-11 protein in thepresence and absence of a test compound and identifying those compoundswhich alter the activity of the CARD-9, CARD-10, or CARD-11 protein.

The invention also features methods for identifying a compound whichmodulates the expression of CARD-9, CARD-10, or CARD-11 by measuring theexpression of CARD-9, CARD-10, or CARD-11 in the presence and absence ofa compound.

In another aspect, the invention features an isolated nucleic acidmolecule comprising a nucleic acid sequence encoding a polypeptidecomprising the amino acid sequence of SEQ ID NO: 8. In one embodiment,the isolated nucleic acid molecule comprises a nucleic acid sequenceencoding a polypeptide consisting of the amino acid sequence of SEQ IDNO: 8.

In another aspect, the invention features an isolated nucleic acidmolecule comprising a nucleic acid sequence encoding a polypeptidecomprising at least 25, e.g., at least 50, 100, 200, 400, 800, 1000, or1100, contiguous amino acids of the amino acid sequence of SEQ ID NO: 8.

In another aspect, the invention features an isolated nucleic acidmolecule comprising the nucleotide sequence of SEQ ID NO: 9. In oneembodiment, the isolated nucleic acid molecule comprises the nucleotidesequence of SEQ ID NO: 7.

In another aspect, the invention features an isolated nucleic acidmolecule comprising at least 50, e.g., at least 100, 200, 400, 800,1200, 1600, 2000, 2500, or 3000, contiguous nucleotides of thenucleotide sequence of SEQ ID NO: 7.

In another aspect, the invention features an isolated nucleic acidmolecule comprising a nucleic acid sequence encoding a fusion proteincomprising amino acids 23-123 of SEQ ID NO: 8. In one embodiment, thenucleic acid molecule comprises a nucleic acid sequence encoding afusion protein comprising the entire sequence of SEQ ID NO: 8.

In another aspect, the invention features an isolated nucleic acidmolecule comprising a nucleotide sequence encoding a polypeptidecomprising an amino acid sequence that is at least 85% identical to theamino acid sequence of SEQ ID NO: 8, wherein percent identity iscalculated using ALIGN program in the GCG software package using aPAM120 weight residue table, a gap length penalty of 12, and a gappenalty of 4.

In another aspect, the invention features an isolated nucleic acidmolecule that hybridizes to a nucleic acid molecule consisting of thenucleotide sequence of SEQ ID NO: 7 under conditions of incubation at45° C. in 6.0×SSC followed by washing in 0.2×SSC/0.1% SDS at 50° C.

In another aspect, the invention features an isolated nucleic acidmolecule that hybridizes to a nucleic acid molecule consisting of thenucleotide sequence of SEQ ID NO: 7 under conditions of incubation at45° C. in 6.0×SSC followed by washing in 0.2×SSC/0.1% SDS at 65° C.

In another aspect, the invention features a nucleic acid describedherein, further comprising vector nucleic acid sequences.

In another aspect, the invention features a host cell, e.g., a mammalianhost cell, containing a nucleic acid described herein.

In another aspect, the invention features an isolated polypeptidecomprising the amino acid sequence of SEQ ID NO: 8. In one embodiment,the polypeptide consists of the amino acid sequence of SEQ ID NO: 8.

In another aspect, the invention features an isolated polypeptidecomprising at least 25, e.g., at least 50, 100, 200, 400, 800, 1000, or1100, contiguous amino acids of the amino acid sequence of SEQ ID NO: 8.

In another aspect, the invention features a fusion protein comprisingamino acids 23-123 of SEQ ID NO: 8. In one embodiment, the fusionprotein comprises the entire sequence of SEQ ID NO: 8.

In another aspect, the invention features an antibody which selectivelybinds to a polypeptide comprising the amino acid sequence of SEQ ID NO:8.

In another aspect, the invention features a method for producing apolypeptide comprising the amino acid sequence of SEQ ID NO: 8, themethod comprising culturing a host cell described herein underconditions in which a polypeptide comprising the amino acid sequence ofSEQ ID NO: 8 is expressed.

In another aspect, the invention features a method for detecting thepresence of a polypeptide in a sample, the method comprising: contactingthe sample with a compound that selectively binds to a polypeptidecomprising the amino acid sequence of SEQ ID NO: 8; and determiningwhether the compound binds to a polypeptide in the sample. In oneembodiment of the method, the compound that selectively binds to thepolypeptide is an antibody.

In another aspect, the invention features a kit comprising a compoundthat selectively binds to a polypeptide comprising the amino acidsequence of SEQ ID NO: 8 and instructions for use. In one embodiment ofthe kit, the compound that selectively binds to the polypeptide is anantibody.

In another aspect, the invention features a method for identifying acompound that binds to a polypeptide, the method comprising the stepsof: contacting a cell or a sample comprising a polypeptide comprisingamino acids 23-123 of SEQ ID NO: 8 with a test compound; and determiningwhether the polypeptide binds to the test compound. In one embodiment,the polypeptide comprises the amino acid sequence of SEQ ID NO: 8.

In another aspect, the invention features a method for identifying acompound that modulates (increases or decreases) the ability of apolypeptide to bind to Bcl-10, the method comprising: contacting apolypeptide comprising amino acids 23-123 of SEQ ID NO: 8 with a testcompound; and determining the effect of the test compound on the abilityof the polypeptide to bind to Bcl-10. In one embodiment, the polypeptidecomprises the amino acid sequence of SEQ ID NO: 8. In general, themethod entails measuring the ability of the polypeptide to bind toBcl-10 in the presence and absence of a test compound or test compounds,and identifying the compound or compounds that modulate the ability ofthe polypeptide to bind to Bcl-10.

In another aspect, the invention features a method for identifying acompound that modulates (increases or decreases) the ability of apolypeptide to increase the activity of NF-kB, the method comprising:contacting a polypeptide comprising amino acids 23-123 of SEQ ID NO: 8with a test compound; and determining the effect of the test compound onthe ability of the polypeptide to increase the activity of NF-kB. In oneembodiment, the polypeptide comprises the amino acid sequence of SEQ IDNO: 8. In general, the method entails measuring the ability of thepolypeptide to increase the activity of NF-kB in the presence andabsence of a test compound or test compounds, and identifying thecompound or compounds that modulate the ability of the polypeptide toincrease the activity of NF-kB.

In another aspect, the invention features a method for detecting thepresence of a nucleic acid molecule in a sample, the method comprising:contacting the sample with a nucleic acid probe or primer whichselectively hybridizes to a nucleic acid molecule comprising SEQ ID NO:7 or SEQ ID NO: 9; and determining whether the nucleic acid probe orprimer binds to a nucleic acid molecule in the sample. In oneembodiment, the sample comprises mRNA molecules and is contacted with anucleic acid probe.

In another aspect, the invention features a method for modulating theactivity of a polypeptide, the method comprising contacting apolypeptide comprising the amino acid sequence of SEQ ID NO: 8 or a cellexpressing the polypeptide with a compound that binds to the polypeptidein a sufficient concentration to modulate the activity of thepolypeptide. In one embodiment, the compound modulates the ability ofthe polypeptide to bind to Bcl-10. In another embodiment, the compoundmodulates the ability of the polypeptide to increase the activity ofNF-kB.

In another aspect, the invention features a method of treating adisorder associated with inappropriate apoptosis, the method comprising:selecting an individual that has a disorder associated withinappropriate apoptosis; and modulating the expression or activity of apolypeptide comprising the amino acid sequence of SEQ ID NO: 8.

In another aspect, the invention features a method of treating adisorder associated with inappropriate lymphocyte activation, the methodcomprising: selecting an individual that has a disorder associated withinappropriate lymphocyte activation; and modulating the expression oractivity of a polypeptide comprising the amino acid sequence of SEQ IDNO: 8.

Other features and advantages of the invention will be apparent from thefollowing detailed description and claims.

BRIEF DESCRIPTION OF THE DRAWINGS

FIGS. 1 A-B depict the cDNA sequence (SEQ ID NO: 1) and the predictedamino acid sequence ( SEQ ID NO: 2) of rat CARD-9. The open readingframe of rat CARD-9 (SEQ ID NO: 1) extends from nucleotide 113 tonucleotide 1720 of SEQ ID NO: 1(SEQ ID NO: 3).

FIG. 2 depicts a hydropathy plot of rat CARD-9. Relatively hydrophobicresidues are above the dashed horizontal line, and relativelyhydrophilic residues are below the dashed horizontal line. The cysteineresidues (cys) and potential N-glycosylation sites (Ngly) are indicatedby short vertical lines just below the hydropathy trace.

FIG. 3 depicts a plot showing the predicted structural features of ratCARD-9. This figure shows the predicted alpha regions (Garnier-Robsonand Chou-Fasman), the predicted beta regions (Garnier-Robson andChou-Fasman), the predicted turn regions (Garnier-Robson andChou-Fasman) and the predicted coil regions (Garnier-Robson). Alsoincluded in the figure is a hydrophilicity plot (Kyte-Doolittle), thepredicted alpha and beta-amphipathic regions (Eisenberg), the predictedflexible regions (Karplus-Schulz), the predicted antigenic index(Jameson-Wolf) and the predicted surface probability plot (Emini).

FIG. 4A depicts an alignment of amino acids 7-98 of rat CARD-9 (aminoacid residues 7-98 of SEQ ID NO: 2) with a consensus caspase recruitmentdomain (CARD) derived from a hidden Markov model.

FIG. 4B depicts an alignment of amino acids 197-213 of rat CARD-9 (aminoacid residues 197-213 of SEQ ID NO: 2) with a consensusindole-3-glycerol phosphate synthase domain derived from a hidden Markovmodel.(SEQ ID NO:14).

FIG. 4C depicts an alignment of amino acids 285-338 of rat CARD-9 (aminoacid residues 285-338 of SEQ ID NO: 2) with a consensus cysteine richrepeat domain derived from a hidden Markov model.(SEQ ID NO: 15).

FIGS. 5A-B depict the cDNA sequence ( SEQ ID NO: 4) and the predictedamino acid sequence ( SEQ ID NO: 5) of human CARD-9. The open readingframe of human CARD-9 ( SEQ ID NO: 4) extends from nucleotide 144 tonucleotide 1751 of SEQ ID NO: 4 ( SEQ ID NO: 6).

FIG. 6 depicts a hydropathy plot of human CARD-9. Relatively hydrophobicresidues are above the dashed horizontal line, and relativelyhydrophilic residues are below the dashed horizontal line. The cysteineresidues (cys) and potential N-glycosylation sites (Ngly) are indicatedby short vertical lines just below the hydropathy trace.

FIG. 7 depicts a plot showing the predicted structural features of humanCARD-9. This figure shows the predicted alpha regions (Garnier-Robsonand Chou-Fasman), the predicted beta regions (Garnier-Robson andChou-Fasman), the predicted turn regions (Garnier-Robson andChou-Fasman) and the predicted coil regions (Garnier-Robson). Alsoincluded in the figure is a hydrophilicity plot (Kyte-Doolittle), thepredicted alpha and beta-amphipathic regions (Eisenberg), the predictedflexible regions (Karplus-Schulz), the predicted antigenic index(Jameson-Wolf) and the predicted surface probability plot (Emini).

FIG. 8A depicts an alignment of amino acids 7-98 of human CARD-9 (aminoacid residues 7-98 of SEQ ID NO: 5) with a consensus caspase recruitmentdomain (CARD) derived from a hidden Markov model.(SEQ ID NO: 13).

FIG. 8B depicts an alignment of amino acids 197-213 of human CARD-9(amino acid residues 197-213 of SEQ ID NO: 5) with a consensusindole-3-glycerol phosphate synthase domain derived from a hidden Markovmodel.(SEQ ID NO: 14).

FIG. 8C depicts an alignment of amino acids 285-338 of human CARD-9(amino acid residues 285-338 of SEQ ID NO: 5) with a consensus cysteinerich repeat domain derived from a hidden Markov model.(SEQ ID NO: 15).

FIG. 9 depicts an alignment of amino acids 1-536 of rat CARD-9 (aminoacid residues 1-536 of SEQ ID NO: 2) and amino acids 1-536 of humanCARD-9 (amino acid residues 1-536 of SEQ ID NO: 5). This alignment wascreated using GAP (gap weight 12; length weight 4). In this alignmentthe sequences are 86.1% identical.

FIGS. 10A-C depict the cDNA sequence ( SEQ ID NO: 7) and the predictedamino acid sequence ( SEQ ID NO: 8) of human CARD-10. The open readingframe of human CARD-10 ( SEQ ID NO: 7) extends from nucleotide 41 tonucleotide 3136 of SEQ ID NO: 7 ( SEQ ID NO: 9).

FIG. 11 depicts a hydropathy plot of human CARD-10. Relativelyhydrophobic residues are above the dashed horizontal line, andrelatively hydrophilic residues are below the dashed horizontal line.The cysteine residues (cys) and potential N-glycosylation sites (Ngly)are indicated by short vertical lines just below the hydropathy trace.

FIG. 12 depicts a plot showing the predicted structural features ofhuman CARD-10. This figure shows the predicted alpha regions(Garnier-Robson and Chou-Fasman), the predicted beta regions(Garnier-Robson and Chou-Fasman), the predicted turn regions(Garnier-Robson and Chou-Fasman) and the predicted coil regions(Garnier-Robson). Also included in the figure is a hydrophilicity plot(Kyte-Doolittle), the predicted alpha and beta-amphipathic regions(Eisenberg), the predicted flexible regions (Karplus-Schulz), thepredicted antigenic index (Jameson-Wolf) and the predicted surfaceprobability plot (Emini).

FIG. 13A depicts an alignment of amino acids 24-115 of human CARD-10(amino acid residues 24-115 of SEQ ID NO: 8) with a consensus caspaserecruitment domain (CARD) derived from a hidden Markov model.(SEQ ID NO:13).

FIG. 13B depicts an alignment of amino acids 366-398 of human CARD-10(amino acid residues 366-398 of SEQ ID NO: 8) with a consensustropomyosin domain derived from a hidden Markov model.(SEQ ID NO: 16).

FIGS. 14A-C depict the cDNA sequence (SEQ ID NO: 10) and the predictedamino acid sequence (SEQ ID NO: 11) of human CARD-11. The open readingframe of human CARD-11 ( SEQ ID NO: 10) extends from nucleotide 328 tonucleotide 3768 of SEQ ID NO:I0 ( SEQ ID NO: 12).

FIG. 15 depicts a hydropathy plot of human CARD-11. Relativelyhydrophobic residues are above the dashed horizontal line, andrelatively hydrophilic residues are below the dashed horizontal line.The cysteine residues (cys) and potential N-glycosylation sites (Ngly)are indicated by short vertical lines just below the hydropathy trace.

FIG. 16 depicts a plot showing the predicted structural features ofhuman CARD-11. This figure shows the predicted alpha regions(Garnier-Robson and Chou-Fasman), the predicted beta regions(Garnier-Robson and Chou-Fasman), the predicted turn regions(Garnier-Robson and Chou-Fasman) and the predicted coil regions(Garnier-Robson). Also included in the figure is a hydrophilicity plot(Kyte-Doolittle), the predicted alpha and beta-amphipathic regions(Eisenberg), the predicted flexible regions (Karplus-Schulz), thepredicted antigenic index (Jameson-Wolf) and the predicted surfaceprobability plot (Emini).

FIG. 17A depicts an alignment of amino acids 12-103 of human CARD-11(amino acid residues 12-103 of SEQ ID NO: 11) with a consensus caspaserecruitment domain (CARD) derived from a hidden Markov model.(SEQ ID NO:13).

FIG. 17B depicts an alignment of amino acids 635-747 of human CARD-11(amino acid residues 635-747 of SEQ ID NO: 11) with a consensus PDZdomain derived from a hidden Markov model.(SEQ ID NO: 17).

FIG. 17C depicts an alignment of amino acids 1003-1091 of human CARD-11(amino acid residues 1003-1091 of SEQ ID NO: 11) with a consensus GUKdomain derived from a hidden Markov model.(SEQ ID NO: 18).

FIGS. 18A, 18B, 18C, 18D, 18E, and 18F depict the results of a series ofexperiments demonstration that CARD-9 interacts directly with Bcl-10. Inthese studies, 293T cells were transfected with expression plasmidsencoding T7 epitope-tagged, Flag epitope-tagged, or Myc-epitope-taggedBcl-10 or CARD-9 as indicated. After 36 h, extracts were prepared andimmunoprecipitated (IP) with a monoclonal antibody to the Flag epitope.The immunoprecipitates were analyzed by SDS-PAGE and immunoblotted witha horseradish peroxidase-conjugated T7-antibody (FIG. 18A) orMYC-antibody (FIGS. 18B and 18C). The cellular extracts were alsoimmunoblotted (WB) with anti-Flag and anti-T7 antibody (upper panel ofFIG. 18A), anti-Flag or anti-Myc antibody (upper and middle panel ofFIG. 18D and FIG. 18C). FIG. 18A demonstrates the in vivo interaction ofCARD-9 with Bcl-10. FIG. 18B and FIG. 18C demonstrate that CARD-9 canself-associate and interact with Bcl-10 through the CARD domain. FIG.18D demonstrates in vitro interaction of CARD-9 with GST-Bcl-10. In thisstudy, ³⁵S-labeled CARD-9 (lane 1, 5% input) was precipitated with anequal amount of glutathione sepharose beads bound to an equal amount ofproteins, GST (lane2), GST-Bcl-10 (lane3), GST-Bcl-10-L41R (lane 4) andthen analyzed by SDS-PAGE and autoradiography. The point mutation L41 Rwithin the CARD domain abrogates its ability to interact with CARD-9.FIG. 18E and FIG. 18F demonstrate the endogenous interaction of CARD-9with Bcl-10. Thp1 cell (5×10⁶) extracts were collected andimmunoprecipitated with a monoclonal antibody to the Bcl-10 epitope(FIG. 18E and FIG. 18F, lane 2), T7 antibody (FIG. 18E and FIG. 18F,lane 5; HC, heavy chain; LC, light chain), or anti-mouse IgG agarose(FIG. 18E, lane 3). The immunoprecipitates were analyzed by SDS-PAGE andimmunoblotted with CARD-9 polyclonal antibody (FIG. 18D) or Bcl-10monoclonal antibody (FIG. 18E). The cellular extracts were alsoimmunoblotted (WB) with polyclonal CARD-9 antibody (FIG. 18E, lane 1) ormonoclonal Bcl-10 antibody (FIG. 18F, lane 1) to detect the endogenousprotein expression. Also, Bcl-10 antibody was incubated with theanti-mouse IgG agarose and analyzed by immunoblotting with CARD-9antibody to rule out the possibility of cross-reaction (FIG. 18E, lane4).

FIG. 19A depicts the results of an experiment demonstrating theconcentration-dependent activation of NF-PB activity by CARD-9. In thisstudy, plasmids expressing CARD-9 were transfected into 293T cells andrelative luciferase activities were determined to measure induction ofNF-PB activity.

FIG. 19B depicts the results of an experiment demonstrating that CARD-9interacts with endogenous Bcl10. In this study, cell extracts wereimmunoprecipitated (IP) with BCl10 antibodies and immunoblotted (WB)with anti-Myc antibodies to detect epitope-tagged CARD-9.

FIG. 19C depicts the results of a functional mapping study of the CARD-9NF-PB-activating domain. In this study, plasmids expressing individualdomains fused to GFP were transfected into 293T cells and induction ofNF-PB activity was measured.

FIG. 20A depicts the concentration-dependent activation of NF-PBactivity by CARD-11.

FIG. 20B is a schematic depiction of deletion mutants of CARD-11 used tomap domains involved in the induction of NF-PB activity. Bars indicatedomains expressed:

-   construct 1 (CARD-11, residues 1-1147), construct 2 (CARD-11,    residues 127-1147),-   construct 3 (CARD-11, residues 1-468), construct 4 (CARD-11,    residues 1-759),-   construct 5 (CARD-11, residues 1-869), construct 6 (CARD-I 1,    residues 469-1147

FIGS. 20C depicts the induction of NF-PB activity by CARD-11 deletionmutants.

FIG. 21 depicts the results of experiments demonstrating that CARD-11interacts with Bcl-10 in mammalian cells. 293T cells were transfectedwith the indicated combinations of expression constructs encodingFlag-Bcl-10, Myc-CARD-11/CARD+CC and Myc-CARD-11/CC. Cell lysates werecollected and immunoprecipitated (IP) and immunoblotted (WB) with eitherFlag or Myc antibodies.

FIG. 22 depicts the results of an experiment demonstrating that CARD-11induces phosphorylation of Bcl-10. 293T cells were transfected withexpression constructs encoding HA-Bcl-10 and either Myc-tagged CARD-11.Cell lysates were collected and immunoblotted (WB) with HA and Mycantibodies to detect Bcl-10 and CARD-11 proteins..

FIG. 23 depicts the results of experiments demonstrating that the CARDdomain of CARD-10 interacts with the CARD domain of Bcl-10 in amammalian two-hybrid assay.

FIG. 24A depicts the results of experiments demonstrating that CARD-10interacts with Bcl-10 in mammalian cells. 293T cells were transfectedwith the indicated CARD-10 and Bcl-10 expression constructs. Cellextracts were immunoprecipitated (IP) with an anti-Bcl-10 antibody andimmunoblotted (WB) with an anti-FLAG antibody to detect epitope taggedCARD-10.

FIG. 24B depicts the results of experiments demonstrating that CARD-10interacts with endogenous Bcl-10 in mammalian cells. 293T cells weretransfected with the indicated CARD-10 expression constructs. Cellextracts were immunoprecipitated (IP) with either anti-Bcl-10 antibodies(lanes 2, 4, and 6) or control T7 monoclonal antibodies (lanes 1, 3, and5) and immunoblotted (WB) with anti-FLAG antibodies to detect epitopetagged CARD-10.

FIG. 25A depicts the results of experiments demonstrating that CARD-10activates NF-PB in a concentration-dependent manner.

FIG. 25B is a schematic depiction of deletion mutants of CARD-10 used tomap domains involved in the induction of NF-PB activity. The barsindicate the domains contained in the constructs: construct 1 (residues1-1032 of SEQ ID NO: 8); construct 2 (residues 131-1032 of SEQ ID NO:8); construct 3 (residues 1-492 of SEQ ID NO: 8); construct 4 (residues1-683 of SEQ ID NO: 8); construct 5 (residues 1-825 of SEQ ID NO: 8);construct 6 (residues 493-1032 of SEQ ID NO: 8); and construct 7(residues 131-432 of SEQ ID NO: 8).

FIG. 25C depicts the results of experiments demonstrating the inductionof NF-PB activity by CARD-10 deletion mutants.

DETAILED DESCRIPTION OF THE INVENTION

The present invention is based, in part, on the identification of apredicted mRNA sequence encoding rat CARD-9 protein. A nucleotidesequence encoding a rat CARD-9 protein is shown in FIGS. 1A-B ( SEQ IDNO: 1; SEQ ID NO: 3 includes the open reading frame only). A predictedamino acid sequence of rat CARD-9 protein is also shown in FIGS. 1A-B (SEQ ID NO: 2).

The present invention is also based, in part, on the identification of apredicted mRNA sequence encoding human CARD-9 protein. A nucleotidesequence encoding a human CARD-9 protein is shown in FIGS. 5A-B ( SEQ IDNO: 4; SEQ ID NO: 6 includes the open reading frame only). A predictedamino acid sequence of human CARD-9 protein is also shown in FIGS. 5A-B( SEQ ID NO: 5).

The present invention is also based, in part, on the identification of apredicted mRNA sequence encoding human CARD-10 protein. A nucleotidesequence encoding a human CARD-10 protein is shown in FIGS. 10A-C ( SEQID NO: 7; SEQ ID NO: 9 includes the open reading frame only). Apredicted amino acid sequence of human CARD-10 protein is also shown inFIGS. 10A-C ( SEQ ID NO: 8).

The present invention is also based, in part, on the identification of apredicted mRNA sequence encoding human CARD-11 protein. A nucleotidesequence encoding a human CARD-11 protein is shown in FIGS. 14A-C (SEQID NO: 10; SEQ ID NO: 12 includes the open reading frame only). Apredicted amino acid sequence of human CARD-11 protein is also shown inFIGS. 14A-C (SEQ ID NO: 11).

Identification of Rat CARD-9

A cDNA encoding rat CARD-9 was identified by a hidden Markov model (HMM)search for CARD-encoding cDNA sequences present in a proprietary ratneuronal cDNA library. The search of the translated cDNA sequencedatabase was performed in three reading frames, forward and reverse.This search led to the identification of a sequence predicted to encodea CARD domain-containing protein later identified as rat CARD-9.

FIGS. 1A-B depict the sequence of a 1879 nucleotide cDNA (SEQ ID NO: 1)which includes a predicted open reading frame ( SEQ ID NO: 3;nucleotides 113-1720 of SEQ ID NO: 1) encoding a 536 amino acid ratCARD-9 protein ( SEQ ID NO: 3). Rat CARD-9 is predicted to be anintracellular protein. This CARD-9 protein is predicted to have amolecular weight of 62.2 kD prior to post-translational modifications.

The predicted amino acid sequence of rat CARD-9 was compared to aminoacid sequences of known proteins and various motifs were identified. The536 amino acid rat CARD-9 protein includes an N-glycosylation site(e.g., about amino acid residues 524-527 of SEQ ID NO: 2); two cAMP- andcGMP-dependent protein kinase phosphorylation sites (e.g., about aminoacid residues 92-95 and 228-231 of SEQ ID NO: 2); nine protein kinase Cphosphorylation sites (e.g., about amino acid residues 16-18, 95-97,138-140, 231-233, 303-305, 362-364, 431-433, 451-453, and 514-516 of SEQID NO: 2); 13 casein kinase II phosphorylation sites (e.g., about aminoacid residues 2-5, 12-15, 23-26, 95-98, 138-141, 171-174, 267-270,362-365, 374-377, 425-428, 483-486, 526-529, and 531-534 of SEQ ID NO:2); a tyrosine kinase phosphorylation site (e.g., about amino acidresidues 176-183 of SEQ ID NO: 2); and an N-myristoylation site (e.g.,about amino acid residues 523-528 of SEQ ID NO: 2).

FIG. 2 depicts a hydropathy plot of rat CARD-9. Relatively hydrophobicresidues are above the dashed horizontal line, and relativelyhydrophilic residues are below the dashed horizontal line. The cysteineresidues (cys) and potential N-glycosylation sites (Ngly) are indicatedby short vertical lines just below the hydropathy trace.

A plot showing the predicted structural features of rat CARD-9 ispresented in FIG. 3. This figure shows the predicted alpha regions(Garnier-Robson and Chou-Fasman), the predicted beta regions(Garnier-Robson and Chou-Fasman), the predicted turn regions(Garnier-Robson and Chou-Fasman) and the predicted coil regions(Garnier-Robson). Also included in the figure is a hydrophilicity plot(Kyte-Doolittle), the predicted alpha and beta-amphipathic regions(Eisenberg), the predicted flexible regions (Karplus-Schulz), thepredicted antigenic index (Jameson-Wolf) and the predicted surfaceprobability plot (Emini).

An analysis of the predicted rat CARD-9 amino acid sequence showed it tocontain several potential functional domains: a CARD domain (e.g., aboutamino acid residues 7-98 of SEQ ID NO: 2); a coiled-coil domain (e.g.,about amino acid residues 140-416 of SEQ ID NO: 2); an indole-3-glycerolphosphate synthase homology region (e.g., about amino acid residues197-213 of SEQ ID NO: 2); and a cysteine rich repeat homology region(e.g., about amino acid residues 285-338 of SEQ ID NO: 2).

FIG. 4A depicts an alignment of amino acids 7-98 of rat CARD-9 (aminoacid residues 7-98 of SEQ ID NO: 2) with a consensus caspase recruitmentdomain (CARD) derived from a HMM.

FIG. 4B depicts an alignment of amino acids 197-213 of rat CARD-9 (aminoacid residues 197-213 of SEQ ID NO: 2) with a consensusindole-3-glycerol phosphate synthase domain derived from a HMM.

FIG. 4C depicts an alignment of amino acids 285-338 of rat CARD-9 (aminoacid residues 285-338 of SEQ ID NO: 2) with a consensus cysteine richrepeat domain derived from a HMM.

The domain alignments depicted in FIGS. 4A-C were identified by homologysearching using consensus domains derived from hidden Markov models(HMMs). HMMs can be used to perform multiple sequence alignment and verysensitive database searching, using statistical descriptions of adomain's consensus sequence. For more information on HMM searches, see,e.g., the HMM website maintained by Washington University.. In thealignments of FIGS. 4A-C a single letter amino acid designation at aposition on the line between the CARD-9 sequence and the HMM-generatedconsensus domain sequence indicates an exact match between the two. A“+” in this middle line indicates a conservative substitution at theparticular residue of CARD-9. Amino acid residues located in the domainsidentified by the HMM search may be important for the appropriatefunctioning of the CARD-9 protein. For this reason, amino acidsubstitutions with respect to the sequence of SEQ ID NO: 2 that areoutside of the domains homologous to HMM consensus domains may be lessdetrimental to the activity of the CARD-9 protein.

Identification of Human CARD-9

A cDNA encoding human CARD-9 was identified by using the rat CARD-9 cDNAdescribed above to conduct a BLAST search of cDNA sequences from aproprietary human megakaryocyte library. This search led to theidentification of a sequence predicted to encode a CARDdomain-containing protein later identified as human CARD-9.

FIGS. 5A-B depict the sequence of a 2098 nucleotide cDNA ( SEQ ID NO: 4)which includes a predicted open reading frame ( SEQ ID NO: 6;nucleotides 144-1751 of SEQ ID NO: 4) encoding a 536 amino acid humanCARD-9 protein ( SEQ ID NO: 5). Human CARD-9 is predicted to be anintracellular protein. This CARD-9 protein is predicted to have amolecular weight of 62.2 kD prior to post-translational modifications.

The predicted amino acid sequence of human CARD-9 was compared to aminoacid sequences of known proteins and various motifs were identified. The536 amino acid human CARD-9 protein includes an N-glycosylation site(e.g., about amino acid residues 524-527 of SEQ ID NO: 5); three cAMP-and cGMP-dependent protein kinase phosphorylation sites (e.g., aboutamino acid residues 92-95, 228-231, and 333-336 of SEQ ID NO: 5); sevenprotein kinase C phosphorylation sites (e.g., about amino acid residues95-97, 138-140, 231-233, 303-305, 431-433, 450-452, and 460-462 of SEQID NO: 5); ten casein kinase II phosphorylation sites (e.g., about aminoacid residues 2-5, 23-26, 95-98, 138-141, 267-270, 363-366, 425-428,483-486, 526-529, and 531-534 of SEQ ID NO: 5); a tyrosine kinasephosphorylation site (e.g., about amino acid residues 176-183 of SEQ IDNO: 5); and three N-myristoylation sites (e.g., about amino acidresidues 453-458, 481-486, and 527-532 of SEQ ID NO: 5).

FIG. 6 depicts a hydropathy plot of human CARD-9. Relatively hydrophobicresidues are above the dashed horizontal line, and relativelyhydrophilic residues are below the dashed horizontal line. The cysteineresidues (cys) and potential N-glycosylation sites (Ngly) are indicatedby short vertical lines just below the hydropathy trace.

A plot showing the predicted structural features of human CARD-9 ispresented in FIG. 7. This figure shows the predicted alpha regions(Garnier-Robson and Chou-Fasman), the predicted beta regions(Garnier-Robson and Chou-Fasman), the predicted turn regions(Garnier-Robson and Chou-Fasman) and the predicted coil regions(Garnier-Robson). Also included in the figure is a hydrophilicity plot(Kyte-Doolittle), the predicted alpha and beta-amphipathic regions(Eisenberg), the predicted flexible regions (Karplus-Schulz), thepredicted antigenic index (Jameson-Wolf) and the predicted surfaceprobability plot (Emini).

An analysis of the predicted human CARD-9 amino acid sequence showed itto contain several potential functional domains: a CARD domain (e.g.,about amino acid residues 7-98 of SEQ ID NO: 5); a coiled-coil domain(e.g., about amino acid residues 140-416 of SEQ ID NO: 5); anindole-3-glycerol phosphate synthase homology region (e.g., about aminoacid residues 197-213 of SEQ ID NO: 5); and a cysteine rich repeathomology region (e.g., about amino acid residues 285-338 of SEQ ID NO:5).

FIG. 8A depicts an alignment of amino acids 7-98 of human CARD-9 (aminoacid residues 7-98 of SEQ ID NO: 5) with a consensus caspase recruitmentdomain (CARD) derived from a HMM.

FIG. 8B depicts an alignment of amino acids 197-213 of human CARD-9(amino acid residues 197-213 of SEQ ID NO: 5) with a consensusindole-3-glycerol phosphate synthase domain derived from a HMM.

FIG. 8C depicts an alignment of amino acids 285-338 of human CARD-9(amino acid residues 285-338 of SEQ ID NO: 5) with a consensus cysteinerich repeat domain derived from a HMM.

The domain alignments of FIGS. 8A-C were identified by homologysearching using consensus domains derived from hidden Markov models(HMMs). HMMs can be used to do multiple sequence alignment and verysensitive database searching, using statistical descriptions of adomain's consensus sequence. For more information on HMM searches, see,e.g., the HMM website maintained by Washington University. In thealignments of FIGS. 8A-C a single letter amino acid designation at aposition on the line between the CARD-9 sequence and the HMM-generatedconsensus domain sequence indicates an exact match between the two. A“+” in this middle line indicates a conservative substitution at theparticular residue of CARD-9. Amino acid residues located in the domainsidentified by the HMM search may be important for the appropriatefunctioning of the CARD-9 protein. For this reason, amino acidsubstitutions with respect to the sequence of SEQ ID NO: 5 that areoutside of the domains homologous to HMM consensus domains may be lessdetrimental to the activity of the CARD-9 protein.

The N-terminal region of CARD-9 (residues 7-98) shares significantsimilarity with CARD motifs found in many apoptosis proteins, includingthose found in Bcl-10/CLAP (29% identity, 44% similarity) and RAIDD (28%identity, 40% similarity).

The central region of CARD-9 (residues 140-420 or residues 140-416)contains heptad repeats characteristic of coiled-coil structures thatfunction in protein oligomerization. The COILS2 (Lupas, 1996, TrendsBiochem. Sci. 21:375) program predicts the existence of at least threecoiled-coil regions with a probability of greater than 80% (residues140-230, 243-277 and 332-419) that are interrupted by regions predictedto have low coiled-coil potential. Correspondingly, BLAST analysis ofthis region showed strong similarity to coiled-coil regions of otherproteins, including myosins and plectins.

Northern blot analysis was performed and a 2.1 -kilobase transcriptcorresponding to CARD-9 was identified in a variety of human adulttissues, including spleen, liver, placenta, lung, PBL and brain. CARD-9was also expressed abundantly in the HL60 cancer cell line and showedsome expression in fetal liver tissue.

FIG. 9 depicts an alignment of amino acids 1-536 of rat CARD-9 (aminoacid residues 1-536 of SEQ ID NO: 2) and amino acids 1-536 of humanCARD-9 (amino acid residues 1-536 of SEQ ID NO: 5). This alignment wascreated using GAP (gap weight 12; length weight 4). In this alignmentthe sequences are 86.1% identical.

Identification of Human CARD-10

A cDNA encoding human CARD-10 was identified by using the rat CARD-9cDNA described above to conduct a BLAST search of cDNA sequences from aproprietary skin library. 3′ RACE was performed on a partial cDNAisolated from the skin library to determine the 3′ sequences of thecDNA. The cDNA sequence was predicted to encode a CARD domain-containingprotein and was later identified as human CARD-10.

FIGS. 10A-C depict the sequence of a 3949 nucleotide cDNA ( SEQ ID NO:7) which includes a predicted open reading frame ( SEQ ID NO: 9;nucleotides 41-3136 of SEQ ID NO: 7) encoding a 1032 amino acid humanCARD-10 protein ( SEQ ID NO: 8). Human CARD-10 is predicted to be anintracellular protein. This CARD-10 protein is predicted to have amolecular weight of 115.9 kD prior to post-translational modifications.

The predicted amino acid sequence of human CARD-10 was compared to aminoacid sequences of known proteins and various motifs were identified. The1032 amino acid human CARD-10 protein includes four N-glycosylationsites (e.g., about amino acid residues 76-79, 472-475, 595-598, and712-715 of SEQ ID NO: 8); a glycosaminoglycan attachment site (e.g.,about amino acid residues 638-641 of SEQ ID NO: 8); 14 protein kinase Cphosphorylation sites (e.g., about amino acid residues 68-70, 78-80,293-295, 313-315, 512-514, 558-560, 603-605, 642-644, 754-756, 782-784,830-832, 868-870, 947-949, and 1022-1024 of SEQ ID NO: 8); 19 caseinkinase II phosphorylation sites (e.g., about amino acid residues 18-21,112-115, 242-245, 293-296, 331-334, 412-415, 438-441, 478-481, 510-513,549-552, 570-573, 681-684, 690-693, 714-717, 748-751, 754-757, 869-872,882-885, and 1028-1031 of SEQ ID NO: 8); two tyrosine kinasephosphorylation sites (e.g., about amino acid residues 201-207 and733-739 of SEQ ID NO: 8); 11 N-myristoylation sites (e.g., about aminoacid residues 15-20, 113-118, 309-314, 487-492, 565-570, 656-661,761-766, 809-814, 893-898, 981-986, and 1021-1026 o SEQ ID NO: 8); twoamidation sites (e.g., about amino acid residues 88-91 and 915-918 ofSEQ ID NO: 8); and two leucine zipper patterns (e.g., about amino acidresidues 230-251 and 426-447 of SEQ ID NO: 8).

FIG. 11 depicts a hydropathy plot of human CARD-10. Relativelyhydrophobic residues are above the dashed horizontal line, andrelatively hydrophilic residues are below the dashed horizontal line.The cysteine residues (cys) and potential N-glycosylation sites (Ngly)are indicated by short vertical lines just below the hydropathy trace.

A plot showing the predicted structural features of human CARD-10 ispresented in FIG. 12. This figure shows the predicted alpha regions(Garnier-Robson and Chou-Fasman), the predicted beta regions(Garnier-Robson and Chou-Fasman), the predicted turn regions(Garnier-Robson and Chou-Fasman) and the predicted coil regions(Garnier-Robson). Also included in the figure is a hydrophilicity plot(Kyte-Doolittle), the predicted alpha and beta-amphipathic regions(Eisenberg), the predicted flexible regions (Karplus-Schulz), thepredicted antigenic index (Jameson-Wolf) and the predicted surfaceprobability plot (Emini).

An analysis of the predicted human CARD-10 amino acid sequence showed itto contain several potential functional domains: a CARD domain (e.g.,about amino acid residues 23-123 of SEQ ID NO: 8); a coiled-coil domain(e.g., about amino acid residues 147-457 of SEQ ID NO: 8); an SH3 domain(e.g., about amino acid residues 704-772 of SEQ ID NO: 8); a guanylatekinase (GUK) domain (e.g., about amino acid residues 830-1032 of SEQ IDNO: 8); and a tropomyosin domain (e.g., about amino acid residues366-398 of SEQ ID NO: 8).

Although the CARD of CARD-10 shows significant similarity to those foundin other CARD family members, it is highly similar to the CARD domainsof CARD-11 (58% identity), CARD-14 (46% identity) and CARD-9 (46%).Adjacent to the N-terminal CARD domain are coiled-coil structures withextensive regions of heptad repeats that function in proteinoligomerization and activation (Lupas, 1996, Trends Biochem. Sci.21:375). The COILS2 program predicts with a probability of greater than70% at least four coiled-coil structures in CARD-10 (e.g., located atabout residues 138-206, 210-256, 263-307, and 326-456 of SEQ ID NO: 8)that are interrupted by regions with a lower coiled-coil potential. ThePDZ/SH3/GUK tripartite structure found at the C-terminus classifiesCARD-10 as a member of the MAGUK family of proteins that function toorganize signaling complexes at plasma membranes.

Northern blot analysis was performed and a 4.4-kilobase transcriptcorresponding to CARD-10 was identified in a variety of human adulttissues, including heart, muscle, kidney, liver, intestine, placenta,and lung. CARD-10 also showed abundant expression in lung, liver, andkidney fetal tissues, as well as in multiple cancer cell lines,including HeLa S3, Chronic Myelogenous Leukemia K562 cells, ColorectalAdenocarcinoma SW480 cells and Lung Carcinoma A549 cells.

FIG. 13A depicts an alignment of amino acids 24-115 of human CARD-10(amino acid residues 24-115 of SEQ ID NO: 8) with a consensus caspaserecruitment domain (CARD) derived from a HMM.

FIG. 13B depicts an alignment of amino acids 366-398 of human CARD-10(amino acid residues 366-398 of SEQ ID NO: 8) with a consensustropomyosin domain derived from a HMM.

The domain alignments of FIGS. 13A-B were identified by homologysearching using consensus domains derived from hidden Markov models(HMMs). HMMs can be used to do multiple sequence alignment and verysensitive database searching, using statistical descriptions of adomain's consensus sequence. For more information on HMM searches, see,e.g., the HMM website maintained by Washington University.In thealignments of FIGS. 13A-B a single letter amino acid designation at aposition on the line between the CARD-10 sequence and the HMM-generatedconsensus domain sequence indicates an exact match between the two. A“+” in this middle line indicates a conservative substitution at theparticular residue of CARD-10. Amino acid residues located in thedomains identified by the HMM search may be important for theappropriate functioning of the CARD-10 protein. For this reason, aminoacid substitutions with respect to the sequence of SEQ ID NO: 8 that areoutside of the domains homologous to HMM consensus domains may be lessdetrimental to the activity of the CARD-10 protein.

The CARD-10 amino acid sequence was used to perform a BLASTP searchagainst the publicly available PROT database (see the BLAST websitemaintained by Washington University). This search identified two clones(GenBank™ Accession Numbers CAB63075 and CAB63076) that are each 99%identical to CARD-10 in a region spanning amino acids 705-1032 ofCARD-10. These clones were generated from bacterial clone contigs ofhuman chromosome 22. This analysis indicates that the gene for CARD-10is located on chromosome 22.

The map position of the CARD-10 gene was determined by performing aBLASTN search using the 3,949 nucleotide CARD-10 sequence of SEQ ID NO:7 against the publicly available High Throughput Genome Sequencing(HTGS) nucleotide database (for information on the HTG database, see theHTGS website at the National Center for Biotechnology information,Bethesda,MD. This search identified clone 889J22, GenBank™ AccessionNumber AL031406, located on human chromosome 22q13.1, with which aportion of the CARD-10 cDNA shares 99% identity over a stretch of 1037nucleotides.

Several diseases or inherited traits map to the same region ofchromosome 22q as the CARD-10 gene, suggesting a potential role forCARD-10 in a disease pathology. These diseases include: spinocerebellarataxia 10 (“SCA10” maps to 22q13 and is characterized by cerebellardysfunction and seizures; OMIM No. 603516); and meningioma 1 (“MN1” mapsto 22q12.3-qter and is characterized by familial and sporadicmeningiomas;OMIM No. 156100). The OMIM (Online Mendelian Inheritance inMan) database is a catalog of human genes and genetic disordersdeveloped for the World Wide Web by the National Center forBiotechnology Information. The database can be found at the OMIM websiteat the National Center for Biotechnology information, Bethesda, MD andcontains textual information, pictures, and reference information. Theentire content of the OMIM reference numbers cited above areincorporated by reference.

Identification of Human CARD-11

A cDNA encoding human CARD-11 was identified by using the rat CARD-9cDNA described above to conduct a BLAST search of cDNA sequences from aproprietary T cell library. A cDNA sequence was predicted to encode aCARD domain-containing protein, later identified as human CARD-11.

FIGS. 14A-C depict the sequence of a 4276 nucleotide cDNA ( SEQ ID NO:10) which includes a predicted open reading frame ( SEQ ID NO: 12;nucleotides 328-3768 of SEQ ID NO: 10) encoding a 1147 amino acid humanCARD-11 protein (SEQ ID NO: 11). Human CARD-11 is predicted to be anintracellular protein. This CARD-11 protein is predicted to have amolecular weight of 132.6 kD prior to post-translational modifications.

The predicted amino acid sequence of human CARD-11 was compared to aminoacid sequences of known proteins and various motifs were identified. The1147 amino acid human CARD-11 protein includes five N-glycosylationsites (e.g., about amino acid residues 241-244, 563-566, 584-587,776-779, and 950-953 of SEQ ID NO: 11); four cAMP- and cGMP-dependentprotein kinase phosphorylation sites (e.g., about amino acid residues106-109, 429-432, 510-513, and 634-637 of SEQ ID NO: 11); 12 proteinkinase C phosphorylation sites (e.g., about amino acid residues 7-9,100-102, 105-107, 243-245, 290-292, 459-461, 508-510, 687-689, 787-789,857-859, 879-881, and 935-937 of SEQ ID NO: 11); 22 casein kinase IIphosphorylation sites (e.g., about amino acid residues 7-10, 100-103,162-165, 168-171, 182-185, 286-289, 378-381, 471-474, 476-479, 578-581,692-695, 725-728, 764-767, 779-782, 816-819, 847-850, 872-875, 897-900,926-929, 1003-1006, 1088-1091, and 1120-1123 of SEQ ID NO: 11); threetyrosine kinase phophorylation sites (e.g., about amino acid residues175-183, 189-195, and 1010-1018 of SEQ ID NO: 11); nine N-myristoylationsites (e.g., about amino acid residues 587-592, 678-683, 698-703,710-715, 761-766, 823-828, 853-858, 917-922, and 1050-1055 of SEQ ID NO:11); and an amidation site (e.g., about amino acid residues 282-285 ofSEQ ID NO: 11).

FIG. 15 depicts a hydropathy plot of human CARD-11. Relativelyhydrophobic residues are above the dashed horizontal line, andrelatively hydrophilic residues are below the dashed horizontal line.The cysteine residues (cys) are indicated by short vertical lines justbelow the hydropathy trace.

A plot showing the predicted structural features of human CARD-11 ispresented in FIG. 16. This figure shows the predicted alpha regions(Garnier-Robson and Chou-Fasman), the predicted beta regions(Garnier-Robson and Chou-Fasman), the predicted turn regions(Garnier-Robson and Chou-Fasman) and the predicted coil regions(Garnier-Robson). Also included in the figure is a hydrophilicity plot(Kyte-Doolittle), the predicted alpha and beta-amphipathic regions(Eisenberg), the predicted flexible regions (Karplus-Schulz), thepredicted antigenic index (Jameson-Wolf) and the predicted surfaceprobability plot (Emini).

An analysis of the predicted human CARD-11 amino acid sequence showed itto contain several potential functional domains: a CARD domain (e.g.,about amino acid residues 6-112 of SEQ ID NO: 11); a coiled-coil domain(e.g., about amino acid residues 130-431 of SEQ ID NO: 11; containingdomains at amino acid residues 130-158 and 165-433 of SEQ ID NO: 1 1); aPDZ domain (e.g., about amino acid residues 635-748 of SEQ ID NO: 11);an SH3 domain (e.g., about amino acid residues 766-834 of SEQ ID NO:11); and a guanylate kinase (GUK) domain (e.g., about amino acidresidues 882-1147 of SEQ ID NO: 11).

Northern blot analysis revealed that CARD-11 is expressed as a4.4-kilobase transcript in a variety of adult tissues, including thymus,spleen, liver and PBLs, but is not significantly expressed in brain,heart, muscle, colon, kidney, intestine, and placenta. Low expression ispresent in lung. CARD-11 also showed abundant expression in specificcancer cell lines, including Promyelocytic Leukemia HL-60 cells, ChronicMyelogenous Leukemia K562 cells, Burkitt's Lymphoma Raji cells andColorectal Adenocarcinoma SW480 cells.

FIG. 17A depicts an alignment of amino acids 12-103 of human CARD-11(amino acid residues 12-103 of SEQ ID NO: 11) with a consensus caspaserecruitment domain (CARD) derived from a HMM.

FIG. 17B depicts an alignment of amino acids 635-747 of human CARD-11(amino acid residues 635-747 of SEQ ID NO: 11) with a consensus PDZdomain derived from a HMM.

FIG. 17C depicts an alignment of amino acids 1003-1091 of human CARD-11(amino acid residues 1003-1091 of SEQ ID NO: 11) with a consensus GUKdomain derived from a HMM.

The domain alignments of FIGS. 17A-C were identified by homologysearching using consensus domains derived from hidden Markov models(HMMs). HMMs can be used to do multiple sequence alignment and verysensitive database searching, using statistical descriptions of adomain's consensus sequence. For more information on HMM searches, see,e.g., the HMM website maintained by Washington University. In thealignments of FIGS. 17A-C asingle letter amino acid designation at aposition on the line between the CARD-11 sequence and the HMM-generatedconsensus domain sequence indicates an exact match between the two. A“+” in this middle line indicates a conservative substitution at theparticular residue of CARD-11. Amino acid residues located in thedomains identified by the HMM search may be important for theappropriate functioning of the CARD-11 protein. For this reason, aminoacid substitutions with respect to the sequence of SEQ ID NO: 11 thatare outside of the domains homologous to HMM consensus domains may beless detrimental to the activity of the CARD-11 protein.

Identification of an Interaction Between CARD-9, CARD-10, and CARD-11and Bcl-10

A mammalian two-hybrid screening assay revealed that CARD-9, CARD-10,and CARD-11 interact with the CARD domain of Bcl-10 (CARD-1).

The Stratagene® Mammalian Two-Hybrid Assay Kit (Stratagene, Inc; LaJolla, Calif.) was used to prepare a vector expressing a protein(Gal4-BD/CARD-9, Gal4-BD/CARD-10, or Gal4-BD/CARD-11) consisting of theDNA binding domain of yeast Gal4 (amino acids 1-147) fused to humanCARD-9, CARD-10, or CARD-11. For a description of the Stratagene®Mammalian Two-Hybrid Assay Kit, see e.g., Hosfield and Chang (1999)Strategies Newsletter 2(2):62-65. In addition, a library of DNAsequences encoding CARD domains was used to create a library ofexpression vectors encoding the murine NF-κB transcriptional activationdomain (amino acids 364-550) fused to a CARD domain (NF-κB-AD/CARD). TheGal4-BD/CARD-9, Gal4-BD/CARD-10, or Gal4-BD/CARD-11 vector, theNF-κB-AD/CARD domain vector library, and a luciferase reporter constructwere introduced into human 293T embryonic kidney cells. If a given CARDdomain expressed fused to the NF-κB transcriptional activation domaininteracts with CARD-9, CARD-10, or CARD-11, the NF-κB transcriptionalactivation domain will be brought into proximity with the promotercontrolling luciferase expression, activating luciferase expression andpermitting detection of the interaction. This analysis revealed thatCARD-9, CARD-10, and CARD-11 interact with the CARD domain of Bcl-10.Interactions between Bcl-10 and CARD-9, CARD-10, and CARD-11 aredescribed in further detail in later sections of this application.

Bcl-10 activates NF-κB and apoptosis. It is thought that these functionsare mediated by its C-terminal domain. The N-terminal CARD domain ofBcl-10 is thought mediate these activation functions upon CARD-CARDbinding with an upstream CARD signaling protein. The finding thatCARD-9, CARD-10, and CARD-11 bind to the CARD domain of Bcl-10 suggeststhat these proteins are upstream signaling proteins that regulate Bcl-10functions (i.e. NF-κB and apoptosis). CARD-9, CARD-10, and CARD-11likely interact with Bcl-10 via their respective CARD domains.Oligomerization of CARD-9, CARD-10, or CARD-11 likely brings about anoligomerization of Bcl-10 following CARD-CARD interactions betweenBcl-10 and CARD-9, CARD-10, or CARD-11. This subsequent oligomerizationof Bcl-10 is expected to result in its activation.

CARD-9, CARD-10, CARD-11, and/or splice variants thereof may be eitherpositive or negative regulators of Bcl-10 function. Thus, these proteinsare potential targets for regulating inflammation, cancer, NF-κBsignaling, and apoptosis in human disease.

Modulation of NF-κB Activity by CARD-10 and CARD-11

The binding of CARD-10 and CARD-11 to Bcl-10 described above suggeststhat CARD-10-Bcl-10 and CARD-11 -Bcl-10 interactions are part of asignaling pathway involved in apoptosis and NF-κB activation. Consistentwith this signal transduction model, CARD-10 and CARD-11 were both shownto be inducers of NF-κB activation. Expression of CARD-10 resulted in a134-fold increase in NF-κB activity, whereas CARD-11 expression resultedin a 24-fold increase in NF-κB activity. To evaluate whether theCARD-10-and CARD-11 -mediated NF-κB activation occurs via a Bcl-10interaction, a Bcl-10 dominant negative deletion mutant (amino acids1-104) was co-expressed with either CARD-10 or CARD-11. The dominantnegative Bcl-10 reduced CARD-10 activation of NF-κB by 35-fold (to 10times above background). CARD-11 activation was reduced by 4-fold (to1.7 times above background). This suggests that CARD-10 and CARD-11 areupstream regulators of Bcl-10-mediated NF-κB activation. Modulation ofNF-κB activity by and CARD-10 and CARD-11, as well as CARD-9, isdescribed in further detail in later sections of this application.

The NF-κB activity assay was performed by co-transfecting an NF-κBreporter plasmid with a construct encoding CARD-10 or CARD-11. In thereporter plasmid, the luciferase gene was placed under the control ofthe NF-κB promoter. Relative luciferase activity was determined at theend of the experiment to assess NF-κB pathway activation by CARD-10 orCARD-11.

CARD-9 Interactions with Bcl-10

Mammalian two-hybrid analysis, in vitro binding assays, and in situbinding assays were used to further study the interaction of CARD-9 andBcl-10. To carry out these studies and additional studies on CARD-9, avariety of plasmid were constructed. Plasmids expressing CARD-9 witheither FLAG or Myc epitopes were constructed by inserting the openreading frame of CARD-9 into expression vectors pFLAG CMV-2, pMYC CMV-2,and pCI (Promega), respectively. Constructs encoding epitope-taggedBcl-10 were described previously (Srinivasula et al. 1999 J. Biol. Chem.274:17946). Plasmids expressing GFP fusions were constructed using pEGFP(Clontech). For mammalian two-hybrid assays, pCMV-CARD-9-CARD/AD andpCMV-CARD-9-CARD/BD plasmids were constructed by inserting the CARDdomain (residues 1-110) of CARD-9 into pCMV-AD and pCMV-BD, respectively(Stratagene). pCMV-CARD/AD and pCMV-CARD/BD plasmids were constructed byinserting individual CARD domains into pCMV-CARD/AD and pCMV-BD,respectively (Stratagene): Bcl-10 (residues 1-104), ARC (residues1-110), RICK (residues 417-540), CARD-4 (residues 1 -119), ASC (residues92-195), caspase-1 (residues I -110), caspase-2 (residues 1-1 22),caspase-4 (residues 1-108), caspase-9 (residues 1-111), caspase-11(residues 1-122), caspase-12 (residues 1-121), IAP-1 (residues 423-543),IAP-2 (residues 450-557), Apaf-1 (residues -1-08) and RAIDD (residues1-108). The Bcl-10 monoclonal antibody (Du et al., 2000) was a gift fromM. Dyer. Affinity purified CARD-9 antibody was raised in rabbitsinjected with a 15-mer peptide (QKGWRQGEEDRENTT; SEQ ID NO: 19)corresponding to residues 512-526 of CARD-9 (Research Genetics).

For the additional mammalian two-hybrid assays, 293T cells in 6-wellplates (35-mm wells) were transfected with the following plasmids: 750ng of pCMV-CARD-9/AD, 750 ng of pCMV-CARD/BD, 250 ng of pFR-Luc fireflyreporter (Stratagene), and 250 ng of pRL-TK renilla reporter (Promega).For NF-?B assays, 293T cells were transfected with the followingplasmids: 900 ng of pNF-kB luciferase reporter (Stratagene), 100 ng ofpRL-TK renilla reporter (Promega) and 1000 ng of indicated expressionplasmids. Cells were harvested 24 h after transfection, and fireflyluciferase activity was determined using the Dual-Luciferase ReporterAssay System (Promega). In addition, renilla luciferase activity wasdetermined and used to normalize transfection efficiencies.

Transfection of 293T cells with plasmids expressing the CARD of CARD-9fused to the transcriptional activation domain of mouse NF-κB(CARD-9-CARD/AD) and the CARD of Bcl-10 fused to the DNA-binding domainof the yeast protein GAL4 (Bcl-10-CARD/BD) activated the mammaliantwo-hybrid reporter plasmid resulting in a 250-fold increase in relativeluciferase activity. Likewise, expression of CARD-9-CARD/BD andBcl-10-CARD/AD increased luciferase activity 75-fold. Co-expression ofCARD-9-CARD with other CARD domains failed to activate luciferaseexpression indicating that the CARD of CARD-9 interacts selectively withthe CARD of Bcl-10.

The in vivo relationship between CARD-9 and Bcl-10 was investigated bycoexpressing Flag-tagged CARD-9 and T7-tagged Bcl-10. Briefly, 293Tcells transfected with plasmids expressing Flag-tagged CARD-9 andT7-tagged Bcl-10 were lysed in 50 mM Tris, pH 7.6, 150 mM NaCl, 0.1%Nonidet P-40 buffer and incubated with anti-FLAG-M2 monoclonal antibody(Eastman Kodak Co) or Bcl-10 monoclonal antibody. The immune complexeswere precipitated with protein G-Sepharose (Amersham Pharmacia Bio) ormouse IgG agarose (Sigma Co.), washed extensively, and then subjected toSDS-polyacrylamide gel electrophoresis and immunoblotted with polyclonalantibodies. Immunoprecipitation of Flag-tagged CARD-9 quantitativelycoprecipitated T7-tagged Bcl-10 (FIG. 18A). This association wasdependant on the N-terminal CARD domain of Bcl-10 since CARD-9 failed tocoprecipitate a variant Bcl-10 with a point mutation (L41R; Srinivasulaet al. 1999 J. Biol. Chem. 274:17946) that disrupts CARD-mediatedhomodimerization (FIG. 18B). In addition, CARD-9 self-associated whenexpressed in cells suggesting that oligomerization may play a role inprotein function (FIG. 18C).

To rule out the possibility that other proteins were necessary for theCARD-9/Bcl-10 interaction, the interaction of radiolabeled CARD-9 withGST-Bcl-10 was studied in vitro using a technique described previously(Ahmad et al. 1997 Cancer Research 57:615-619). Briefly Bcl-10 WT andL41R mutant were expressed in DH5 alpha bacteria as GST fusion proteinsand equal amount of proteins were immobilized on glutathione-sepharose(Amersham-Pharmacia). Equal amount of S³⁵ methionine-labeled CARD-9protein was incubated with the protein bound sepharose beads in 100 μlof binding buffer (50 mM Tris-Cl pH 7.6, 120 mM NaCl, 0.5% Brij andprotease inhibitors) for 3 hours. The beads were washed 4 times with thesame buffer and boiled in SDS sample buffer. The proteins were thenresolved on a 10% SDS gel and visualized by autoradiography. Thisanalysis revealed that CARD-9 associates directly with Bcl-10 (FIG.18D). The amount of CARD-9 that associated with GST-Bcl-10 (L41R) wasgreatly reduced confirming the importance of the Bcl-10 CARD domain inmediating interactions between these two proteins.

Components of signaling pathways are frequently found pre-assembledtogether within the cell to ensure a rapid response to upstream stimuli.To examine the interactions between endogenous CARD-9 and Bcl-10, apolyclonal antibody that specifically recognizes CARD-9 was used.Immunoblot analysis of extracts derived from human monocyte Thp1 cellsrevealed a predominant band of approximately 70 kDa corresponding toendogenous CARD-9 (FIG. 18E, lane 1), and a 40 kDa band corresponding toendogenous Bcl-10 (FIG. 18F, lane 1). Immunoprecipitation of Bcl-10coprecipitated CARD-9 indicating that both endogenous proteins areassociated with each other within Thp1 cells (FIG. 18E, lane 2).

To determine the cellular localization of CARD-9, and to confirm thatCARD-9 associated intracellularly with Bcl-10 when the two proteins wereco-expressed, Rat-1 cells were transfected with a Myc-tagged vectorencoding full-length CARD-9, and with an HA-tagged vector encodingfull-length Bcl-10. The expressed proteins were detected using a mixtureof a monoclonal anti-Myc antibody and a polyclonal rabbit anti-HAantibody. Briefly, Rat-1 cells were transfected in glass chamber slides(BioCoat, Becton-Dickinson Labware) with pCI-Bcl-10-HA orpCI-Card-9-myc, alone or together, using FuGENE-6 (Roche MolecularBiochemicals) for 20 hours. Cells were fixed in 4% paraformaldehyde,permeabilized and blocked in buffer containing 0.4% Trition X-100, andsequentially incubated with primary and secondary antibodies. Antibodieswere rabbit anti-HA polyclonal Y-11 (Santa Cruz Biotechnology), mouseanti-myc monoclonal 9E10 (Oncogene Research Products), Alexa-488 Goatanti-mouse IgG (Molecular Probes) and Texas-Red Goat anti-rabbit IgG(Jackson Immunoresearch Laboratories). No cross-reactivity was observedbetween any of the antibodies. Nuclei were stained with Hoechst 33258.Images were acquired using a Nikon E800 microscope with a 60×(oil)objective and Spot digital camera (Diagnostic Instruments, Inc.) drivenby MetaMorph software (Universal Imaging Corp.) with final imagesprepared using Adobe PhotoShop.

The two proteins exhibited distinctly different patterns of cellularlocalization when either vector was transfected alone. Whereas Bcl-10exhibited either a clear pattern of discrete cytoplasmic filaments, or adiffuse whole-cell distribution, CARD-9 displayed a somewhat punctatecytoplasmic or whole-cell distribution, but was not observed to formfilament-like structures. When the two proteins were co-expressed in thesame cell, however, some of the CARD-9 was found to co-localize with theBcl-10 filaments. This finding is consistent with the interaction ofCARD-9 with Bcl-10 observed in co-immunoprecipitation experiments, andsuggests that CARD-9 is recruited to a cytoplasmic signaling complexwith Bcl-10.

Modulation of NF-κB Activity by CARD-9

The ability of CARD-9 to activate NF-κB was investigated using aluciferase reporter gene directed by an NF-κB-responsive promoter.Expression of CARD-9 in 293T cells induced NF-κB activity 8-foldcompared to empty vector (FIG. 19A). Activation was specific for NF-κBsignaling since CARD-9 failed to activate a luciferase reporter genewith AP-1 promoter elements. Activation of NF-κB by CARD-9 correlatedwith binding to endogenous Bcl-10 suggesting that CARD-9 forms aCARD-9/Bcl-10 signaling complex within the transfected cells (FIG. 19B).The domains of CARD-9 that mediate the activation of NF-κB wereinvestigated (FIG. 19C). Since individual domains of CARD-9 were eitherunstable or insoluble, the CARD (residues 1-98), coiled-coil (residues140-418) and C-terminal (residues 99-536) domains of CARD-9 were fusedto GFP and expressed the fusion proteins in 293T cells. The CARD-GFPfusion activated NF-κB signaling to levels similar to that obtained withCARD-9-GFP. The coiled-coil and C-terminal domains when fused to GFPfailed to activate reporter gene expression establishing the CARD domainas the NF-κB activating domain of CARD-9.

These results, taken with the finding of a direct interaction betweenCARD-9 and Bcl-10 suggest that CARD-9 is a specific regulator of Bcl-10function. CARD-9 could play a role as an upstream signaling moleculethat recruits Bcl-10 through CARD/CARD interactions. The resultingsignaling complex may interact directly or indirectly with components ofthe IKK complex resulting in its activation, e.g., througholigomerization of IKKγ. Indeed the data described above data shows thatboth CARD-9 and Bcl-10 form large oligomeric complexes (filaments) whenoverexpressed in mammalian cells. Furthermore, enforced oligomerizationof the C-terminus of Bcl-10/CLAP is thought to induce NF-κB activation,suggesting that the CARD domain of Bcl-10 functions as anoligomerization domain that transduces the activation signal to the IKKcomplex through its C-terminal domain. The ability of CARD-9 to form acomplex with Bcl-10 via CARD/CARD interactions supports the idea thatBcl-10 functions as an adaptor between the effector IKK complex and theproximal signaling complexes that interact with CARD-9. Signalingmolecules upstream of CARD-9 are predicted to transduce their signals toBcl-10 through direct interactions with the C-terminal coiled-coildomain of CARD-9. Taken together, these results identify CARD-9 as animportant mediator of NF-κB signaling through Bcl-10.

Modulation of NF-κB Activity by CARD-11

Studies on the activation of NF-kB activity by CARD-11 were performed asfollows. For NF-kB assays, 293T cells were transfected with thefollowing plasmids: 900 ng of pNF-kB luciferase reporter (Stratagene),100 ng of pRL-TK renilla reporter (Promega) and 1000 ng of indicatedexpression plasmids. Cells were harvested 24 h after transfection, andfirefly luciferase activity was determined using the Dual-LuciferaseReporter Assay System (Promega). In addition, renilla luciferaseactivity was determined and used to normalize transfection efficiencies.

These studies showed that when CARD-11 is expressed in 293T cells, NF-kBactivity is induced 20-to 40-fold compared to empty vector (FIG. 20A).NF-kB signaling occurred through the IKK complex since dominant-negativeversions of IKK-g and IKK-b blocked the ability of CARD-11 to induceNF-kB activity (data not shown). To determine the role of individualdomains in NF-kB signaling, a series of N- and C-terminal truncationmutants of CARD-11 were constructed (FIG. 20B). The N-terminal CARD ofCARD-11 was essential for NF-kB signaling since deletion of this domaineliminated the induction of NF-kB activity (FIG. 20C). Immunoblotanalysis revealed that the mutant proteins were expressed at levelssimilar to wt protein indicating that loss of function was not due toreduced levels of expression. In contrast, the C-terminal PDZ, SH3 andGUK domains were not required for NF-kB signaling since deletion ofthese domains had no effect on the ability of CARD-11 to induce NF-kBactivity. However, a CARD-11 mutant lacking its C-terminal PDZ, SH3 andGUK domains induced NF-kB activity to levels 4-to 5-fold greater thanthat obtained with wt protein (FIG. 20C). Thus, the C-terminal domainsmay function to negatively regulate induction of NF-kB signaling byCARD-11.

CARD-11 Interactions with Bcl-10

To identify the binding partners of CARD-11, a mammalian two-hybridanalysis was performed using the CARD domains of 15 known proteins. Formammalian two-hybrid assays, 293T cells in 6-well plates (35-mm wells)were transfected with the following plasmids: 750 ng of pCMV-CARD-11/AD,750 ng of pCMV-BD fused to individual CARD domains, 250 ng of pFR-Lucfirefly reporter (Stratagene), and 250 ng of pRL-TK renilla reporter(Promega). pCMV-CARD-11 /AD and pCMV-CARD/BD plasmids were constructedby inserting individual CARD domains into pCMV-CARD/AD and pCMV-BD,respectively (Stratagene): Bcl-10 (residues 1-104), ARC (residues1-110), RICK (residues 417-540), CARD-4 (residues 1-1 19), ASC (residues92-195), caspase-I (residues 1-110), caspase-2 (residues 1-122),caspase-4 (residues 1-108), caspase-9 (residues 1-111), caspase-11(residues 1-122), caspase-12 (residues 1-121), IAP-1 (residues 423-543),IAP-2 (residues 450-557), Apaf-1 (residues 1-108) and RAIDD (residues 1-108). Cells were harvested 24 hours after transfection, and fireflyluciferase activity was determined using the Dual-Luciferase ReporterAssay System (Promega). In addition, renilla luciferase activity wasdetermined and used to normalize transfection efficiencies.

The CARD of CARD-11 interacted with the CARD of Bcl10 resulting in a17-fold increase in relative luciferase activity. Co-expression ofCARD-11-CARD with other CARD domains failed to activate luciferaseexpression indicating that the CARD of CARD-11interacts selectively withthe CARD of Bcl-10. These data suggest that CARD-11 is a signalingpartner of Bcl-10.

The interaction between CARD-11 and Bcl-10 was next examined in cells byoverexpressing CARD-11 is cells and performing co-immunoprecipitationassays. These assays were performed as follows. Plasmids expressingCARD-11 with C-terminal Myc epitopes were constructed using pCMV-Tag 5A(Stratagene Corp., La Jolla, Calif.). Constructs encoding epitope-taggedBcl-10 were described previously (Srinivasula et al., 1991 J. Biol.Chem. 274:17946). 293T cells transfected with the plasmids were lysed in50 mM Tris, pH 8.0, 120 mM NaCl, 1 mM EDTA, 0.5% Nonidet P-40 buffer andincubated with anti-Flag-M2 monoclonal antibody (Sigma Co.). The immunecomplexes were precipitated with protein G-Sepharose (Amersham PharmaciaBio), washed extensively, and then subjected to SDS-polyacrylamide gelelectrophoresis and immunoblotted with polyclonal anti-Myc antibodies(Santa Cruz Biotechnology, Inc.). This study showed that Flag-taggedBcl-10 quantitatively co-precipitated Myc-tagged CARD-11 (FIG. 21, lane3). The association is mediated by the N-terminal CARD domain of CARD-11as demonstrated by the fact that Bcl-10 did not co-precipitate with atruncated form of CARD11 lacking its CARD (FIG. 21, lane 5).

To confirm the interaction between CARD-11 and Bcl-10, co-localizationexperiments were performed with full-length CARD-11. In these studies,cells were transfected in glass chamber slides (BioCoat,Becton-Dickinson Labware) with plasmids expressing HA-tagged Bcl10 andMyc-tagged CARD-11 using FuGENE-6 (Roche Molecular Biochemicals) for 20h. Cells were fixed in 4% paraformaldehyde, permeabilized and blocked inbuffer containing 0.4% Trition X -100, and sequentially incubated withprimary and secondary antibodies: rabbit anti-HA polyclonal Y-11 (SantaCruz Biotechnology), mouse anti-Myc monoclonal 9E10 (Oncogene ResearchProducts), Alexa-488 Goat anti-mouse IgG (Molecular Probes) andAlexa-594 Goat anti-rabbit IgG (molecular probes). No cross-reactivitywas observed between any of the antibodies. Images were acquired using aNikon T200 microscope with a 60×oil objective and an Orca100 digitalcamera (Hammamatsu, Inc.) driven by MetaMorph software (UniversalImaging Corp.). Final images were prepared using Adobe PhotoShop.

When these two proteins were co-expressed in the same cell, some of theCARD-11 was found to co-localize with the Bcl-10. This finding isconsistent with an intracellular interaction between CARD-11 and Bcl-10,and suggests that CARD-11 may be recruited to a cytoplasmic signalingcomplex with Bcl10. To test whether the CARD domain of CARD-11 wasrequired for this interaction, the localization of a CARD-11 truncationmutant lacking the N-terminal CARD (CARD-11/DCARD) was examined. Whenexpressed alone, CARD-11/DCARD displayed a similar cellular localizationpattern to that of full-length CARD-11. When co-expressed with Bcl-10,however, CARD-11 /DCARD was not found to co-localize with Bcl-10.

Given that the C-terminal PDZ/SH3/GUK domain of CARD-11 is inhibitory inNF-kB assays, this domain might interfere with the interaction betweenCARD-11 and Bcl10. To test this idea, a CARD-11 protein lacking itsC-terminal domain (CARD11/CARD+CC) was expressed. CARD11/CARD+CC wasmainly localized to the cytoplasm when expressed alone. Whenco-expressed with Bcl-10, however, some of the CARD-11 /CARD+CC wasfound in cytoplasmic aggregates that co-localized with Bcl-10. Deletionof the CARD domain of this vector abolished co-localization the Bcl10cytoplasmic aggregates.

Bcl-10 migrates in SDS gels as a triplet ranging in size from 29 to 32kDa due to phosphorylation of its C-terminal domain (Srinivasula et al.1999 J. Biol. Chem. 274:17946; Koseki et al., 1999 J. Biol. Chem.274:9955-61). Treatment of cell lysates with calf intestinal alkalinephosphatase eliminates the slower migrating forms demonstrating that thefastest migrating band represents unphosphorylated Bcl-10. Sincephosphorylation can play a critical role in signal transduction, studieswere performed to determine whether co-expression of CARD-11 induces thephosphorylation of Bcl-10 (FIG. 22). When expressed alone, HA-taggedBcl-10 is primarily unphosphorylated (lane 1, lower band). However,co-expression of CARD-11 markedly increased the amount of phosphorylatedBcl-10 represented by the slower migrating bands (lane 3 and 8, middleand upper bands). Co-expression of the NF-kB activator CARD-4 did notinduce phosphorylation of Bcl-10 indicating that the observed increasein phosphorylated Bcl-10 is specific to co-expression with CARD 11 (datanot shown). The induction of Bcl-10 phosphorylation is dependent on theN-terminal CARD of CARD-11 since co-expression of truncated mutantslacking these domains has no effect on Bcl-10 phosphorylation levels(lane 5 and 10). Immunoblot analysis revealed that the Myc-taggedtruncation mutants were expressed at levels similar to wt proteinsuggesting that loss of function is not due to reduced levels ofexpression. Taken together, these data suggest that CARD-11 inducesphosphorylation of Bcl-10 via its N-terminal CARD domain.

CARD-11 is a specific regulator of Bcl-10 function. The finding thatCARD-11 binds to Bcl-10 through a CARD/CARD interaction suggests thatthis molecule functions as upstream activator of Bcl-10. As discussedabove, CARD-9 also binds to the CARD activation domain of Bcl-10 andsignals NF-kB activation. Thus, CARD11 and CARD-9 constitute a subclassof CARD proteins that may function to transduce upstream stimuli to theactivation of Bcl-10 and NF-kB. In response to upstream signals, thecoiled-coil domains could mediate self-association of CARD-11 resultingin the aggregation and activation of Bcl-10. Bcl-10 might then engageand oligomerize IKKg resulting in the activation of the IKK complex andNF-kB (Inohara et al. 1999 J. Biol. Chem. 274:14566; Poyet et al.,1999). Thus, CARD-11 could function in a manner analogous to Apaf-1 andCARD-4 that function as upstream regulators to induce oligomerizationand activation of their respective downstream CARD binding partners. Thedata showing that CARD-11 induces the phosphorylation of Bcl-10 suggeststhat signal transduction may involve the participation of aserine/threonine kinase. The C-terminal PDZ/SH3/GUK domains of CARD-11may function in an analogous manner to the C-terminal LRR domain ofCARD-4 and the WD-40 domain of Apaf-1 to regulate protein activation byupstream signals. PDZ/SH3/GUK domains identify MAGUK family members, aclass of proteins that associate with the plasma membrane (Fanning andAnderson, 1999 Curr Opin Cell Biol 11:432-9). Interestingly, the PDZdomain found in many MAGUK proteins has been shown to interact with theintracellular domains of specific receptors. Thus, CARD-11 may functionas a scaffolding protein to assemble a multi-protein complex at theintracellular domain of a receptor that signals the activation of NF-kB.

CARD-10 Interactions with Bcl-10

Mammalian two-hybrid analysis was performed to screen a collection ofCARD domains for selective interaction with the CARD of CARD-10. TheCARD of CARD-10 interacted with the CARD of Bcl-10, resulting in a340-fold increase in relative luciferase activity (FIG. 23).Co-expression of the CARD-10 CARD with 20 other CARDs failed to activateluciferase expression, indicating that the CARD of CARD-10 interactsselectively with the CARD of Bcl-10 (FIG. 23).

The mammalian two-hybrid analysis was performed as follows. 293T cellswere transfected with the mammalian two-hybrid reporter constructpFR-Luc (Stratagene) and the CARD of CARD-10 fused to the binding domainwas screened against a panel of individual CARDs fused to theDNA-binding domain. After 24 hours, cells were collected and assayed forrelative luciferase activity as a measure or protein-proteininteraction. The pCMV-CARD10-CARD/BD plasmids were constructed byinserting the CARD domain of CARD-10 (residues 1-138 of SEQ ID NO: 8)into pCMV-BD (Stratagene). The panel of CARD domains used for themammalian two-hybrid screen was described previously (Bertin et al. 2000J. Biol. Chem. 275:41082). 293T cells in 6-well plates (35-mm wells)were transfected with the following plasmids: 750 ng of pCMV-CARD-10/BD;750 ng of pCMV-AD fused to individual CARD domains; 250 ng of pFR-Lucfirefly reporter (Stratagene); and 250 ng of pRL-TK renilla reporter(Promega). Cells were harvested 24 hours after transfection, and fireflyluciferase activity was determined using the Dual-Luciferase ReporterAssay System (Promega). In addition, renilla luciferase activity wasdetermined and used to normalize transfection efficiencies.

The finding that the CARD of CARD-10 selectively interacts with the CARDof Bcl-10 by mammalian two-hybrid analysis suggested that CARD-10 may bea signaling partner of Bcl-10. Interactions between these two proteins,when overexpressed in cells, were therefore examined. Plasmidsexpressing CARD-10 with C-terminal FLAG epitopes were constructed usingpCMV-Tag 4A (Stratagene). Constructs encoding epitope-tagged Bcl-10 weredescribed previously (Srinivasula et al. 1999 J. Biol. Chem. 274:17946).

Immunoprecipitation of T7-tagged Bcl-10 quantitatively co-precipitatedFLAG-tagged CARD-10 (FIG. 24A, lane 1). This interaction was dependenton the CARDs of both proteins. CARD-10 failed to associate with avariant Bcl-10 with a point mutation (L41R; Srinivasula et al. 1999 J.Biol. Chem. 274:17946) that disrupts CARD-mediated homodimerization(FIG. 24A, lane 3). In addition, a CARD-10 truncation mutant lacking itsCARD domain failed to co-precipitate with Bcl-10 (FIG. 24A, lane 2) orthe Bcl-10 L41R mutant (FIG. 24A, lane 4).

CARD-10, when overexpressed in cells, was shown to interact withendogenous Bcl-10 (FIG. 24B). Endogenous Bcl-10 immunoprecipitatedFLAG-CARD-10 (FIG. 24B, lane 2), but not FLAG-CARD10/ΔCARD (FIG. 24B,lane 4). Immunoprecipitation with Bcl-10 antibodies is depicted in lanes2, 4, and 6 of FIG. 24B. Immunoprecipitation with T7 control antibodiesis depicted in lanes 1, 3 and 5 of FIG. 24B.

In the experiments described above, cells were lysed in 50 mM Tris, pH8.0, 120 mM NaCl, 1 mM EDTA, 0.5% Nonidet P-40 buffer and incubated withindicated antibodies. The immune complexes were precipitated withprotein G-Sepharose (Amersham Pharmacia Bio), washed extensively, andthen subjected to SDS-polyacrylamide gel electrophoresis andimmunoblotted with polyclonal anti-FLAG (Santa Cruz Biotechnology, Inc.)or anti-Bcl-10 antibodies.

Modulation of NF-κB Activity by CARD-10

The ability of CARD-10 to induce NF-κB activity was evaluated by using aluciferase reporter gene assay. When CARD-10 was expressed in 293Tcells, NF-κB activity was induced 90-fold as compared to empty vector,in a CARD-10 concentration-dependent manner (FIG. 25A). Induction ofNF-κB activity was dependent on the IKK complex, since dominant-negativeversions of IKK-γ and IKK-β blocked the ability of CARD-10 to induce theactivation of NF-κB.

To determine the role of individual CARD-10 domains in NF-κB signaling,N-and C-terminal truncation mutants of CARD-10 were constructed (FIG.25B). The N-terminal CARD domain of CARD-10 was essential for NF-κBsignaling, since deletion of this domain eliminated the induction ofNF-kB activity (FIG. 25C, lanes 2, 6, and 7). Immunoblot analysisrevealed that the truncation mutants were expressed at a levelcomparable to the wild type protein. Deletion of the C-terminal GUKdomain did not interfere with the ability of CARD-10 to induce NF-κBactivity (FIG. 25C, lane 5). However, further deletion of the SH3 andPDZ domains reduced the levels of NF-κB activity to levels 2 to 3-foldless than that obtained with wild type protein (FIG. 25C, lanes 3 and4). Immunoblot analysis revealed that the C-terminal truncated proteinswere expressed at levels similar to each other and the wild type protein(FIG. 25C, upper panel), indicating that reductions in activity were notdue to reduced levels of expression. Thus, the PDZ and SH3 domainsappear to be required for maximal activation of NF-kB activity byCARD-10.

For the NF-κB assays described above, 293T cells were transfected withthe following plasmids: 900 ng of pNF-κB luciferase reporter(Stratagene); 100 ng of pRL-TK renilla reporter (Promega); and 1000 ngof indicated expression plasmids. Cells were harvested 24 hours aftertransfection, and firefly luciferase activity was determined using theDual-Luciferase Reporter Assay System (Promega). In addition, renillaluciferase activity was determined and used to normalize transfectionefficiencies.

The finding that CARD-10 both binds to Bcl-10 and signals NF-κBactivation through its N-terminal CARD domain suggests that CARD-10functions as an upstream activator of Bcl-10. CARD-10 is one of fourCARD proteins identified thus far that assemble together with Bcl-10 andsignal the activation of NF-κB (Bertin et al. 2000 J. Biol. Chem.275:41082). These molecules (CARD-10, CARD-9, CARD-11, and CARD-14)likely function to transduce distinct upstream stimuli to the activationof Bcl-10 and NF-κB. This subclass of CARD proteins are related in bothsequence and structure. In addition to containing closely relatedN-terminal CARDs that interact specifically with Bcl-10, each moleculecontains a coiled-coiled domain that could mediate self-associationresulting in aggregation and activation of Bcl-10 in response toupstream signals. Bcl-10 might then engage and oligomerize IKKγresulting in the activation of the IKK complex and NF-κB (Poyet et al.2000 J. Biol. Chem. 275:37966; Inohara et al. 2000 J. Biol. Chem.275:27823). Thus, CARD-10 and the other Bcl-10 activators (e.g., CARD-9,CARD-11 and CARD-14) likely function in a manner analogous to Apaf-1 andCARD-4, molecules that induce oligomerization and activation of theirrespective downstream CARD-binding partners. CARD-10, CARD-11, andCARD-14 each contain a C-terminal PDZ/SH3/GUK domain, the presence ofwhich suggests a role for these proteins in signal transduction byreceptors at the plasma membrane. A recent study implicating Bcl-10 as amediator of antigen receptor signaling in B and T cells suggests thatCARD-10 and the other CARD/MAGUK family members might function torecruit Bcl-10 to receptor complexes. For example, signaling complexesat the plasma membrane (e.g., T and B cell receptors) may recruit andactivate the CARD/MAGUK proteins (CARD-10, CARD-11, and CARD-14) throughtheir C-terminal PDZ/SH3/GUK domains. Bcl-10 might then engage andoligomerize IKKγ resulting in the activation of the IKK complex andNF-κB.

TABLE 1 Summary of Rat CARD-9, Human CARD-9, Human CARD-10, and HumanCARD-11 Sequence Information Gene cDNA Protein ORF Figure Rat SEQ IDNO:1 SEQ ID NO:2 SEQ ID NO:3 FIGS. CARD-9  1A-B Human SEQ ID NO:4 SEQ IDNO:5 SEQ ID NO:6 FIGS. CARD-9  5A-B Human SEQ ID NO:7 SEQ ID NO:8 SEQ IDNO:9 FIGS. CARD-10 10A-C Human SEQ ID NO:10 SEQ ID NO:11 SEQ ID NO:12FIGS. CARD-11 14A-C

TABLE 2 Summary of Domains of Rat CARD-9 Domain Location CARD aboutamino acid residues 7-98 of SEQ ID NO:2 Coiled-coil about amino acidresidues 140-416 of SEQ ID NO:2 Indole-3-glycerol about amino acidresidues 197-213 of SEQ ID NO:2 phosphate synthase Cysteine rich repeatabout amino acid residues 285-338 of SEQ ID NO:2

TABLE 3 Summary of Domains of Human CARD-9 Domain Location CARD aboutamino acid residues 7-98 of SEQ ID NO:5 Coiled-coil about amino acidresidues 140-416 of SEQ ID NO:5 Indole-3-glycerol about amino acidresidues 197-213 of SEQ ID NO:5 phosphate synthase Cysteine rich repeatabout amino acid residues 285-338 of SEQ ID NO:5

TABLE 4 Summary of Domains of Human CARD-10 Domain Location CARD aboutamino acid residues 23-123 of SEQ ID NO:8 Coiled-coil about amino acidresidues 147-457 of SEQ ID NO:8 SH3 about amino acid residues 704-772 ofSEQ ID NO:8 Guanylate about amino acid residues 830-1032 of kinase (GUK)SEQ ID NO:8 tropomyosin about amino acid residues 366-398 of SEQ ID NO:8MAGUK about amino acid residues 457-1032 of SEQ ID NO:8

TABLE 5 Summary of Domains of Human CARD-11 Domain Location CARD aboutamino acid residues 6-112 of SEQ ID NO:11 Coiled-coil about amino acidresidues 130-431 of SEQ ID NO:11 PDZ about amino acid residues 635-748of SEQ ID NO:11 SH3 about amino acid residues 766-834 of SEQ ID NO:11Guanylate about amino acid residues 882-1147 of kinase (GUK) SEQ IDNO:11 MAGUK about amino acid residues 635-1147 of SEQ ID NO:11

A region, the CARD domain, of rat CARD-9 (amino acid residues 7-98),human CARD-9 (amino acid residues 7-98), human CARD-10 (amino acidresidues 23-123), and human CARD-11 (amino acid residues 6-112) proteinsbears some similarity to the CARD domains of CARD-3, CARD-4, CARD-5,CARD-6, CARD-7, CARD-8, CARD-12, CARD-13, CARD-14, and CARD-15. Detailedinformation concerning CARD-3, CARD-4, CARD-5, CARD-6, CARD-7, CARD-8,and CARD-12 can be found in U.S. application Ser. No. 09/245,281, filedFeb. 5, 1999, U.S. application Ser. No. 09/207,359, filed Dec. 8, 1998,U.S. application Ser. No. 09/099,041, filed Jun. 17, 1998, U.S.application Ser. No. 09/019,942, filed Feb. 6, 1998, U.S. applicationSer. No. 09/428,252, filed Oct. 27, 1999, and U.S. application Ser. No.60/161,822, filed Oct. 27, 1999. The entire content of each of theseapplications is incorporated herein by reference. Detailed informationconcerning CARD-13 and CARD-15 can be found in U.S. application Ser. No.09/573,640, filed May 17, 2000. Detailed information concerning CARD-14can be found in U.S. application Ser. No. 60/181,159, filed Feb. 9,2000, and U.S. patent application Ser. No. 09/767,215, filed Jan. 22,2001. The entire content of these applications is incorporated herein byreference.

Membrane-Associated Protein Interactions

Protein-protein interactions are required for the proper assembly ofmembrane-associated protein complexes and for the localization of thesecomplexes to the appropriate membrane domain. These membrane-associatedcomplexes can include, e.g., transmembrane receptors, ion channels, celladhesion molecules, and cytosolic signaling elements. A class ofproteins known as membrane-associated guanylate kinases (MAGUKs) play animportant role in coupling the activity of transmembrane receptors todownstream signaling molecules. Members of the MAGUK protein familycontain a PDZ domain, an SH3 domain, and a guanylate kinase (GUK)domain. MAGUK proteins have been found to be associated with the plasmamembrane, including the discrete focal structures that comprise thehighly ordered synapses. Studies of MAGUKs suggest that they function asscaffolding proteins and that the PDZ domains are used to tethertransmembrane proteins in specific structural domains within the plasmamembrane (Fanning and Anderson (1999) Current Opinion in Cell Biology11:432).

A C-terminal region of human CARD-11 (about amino acid residues 635-1147of SEQ ID NO: 11) and CARD-10 (about amino acid residues 457-1032 of SEQID NO: 8) contains a series of domains found in members of the MAGUKprotein family. All three of the domains of MAGUK family members arepresent in CARD-11: a PDZ domain (e.g., about amino acid residues635-748 of SEQ ID NO: 11); an SH3 domain (e.g., about amino acidresidues 766-834 of SEQ ID NO: 11); and a GUK domain (e.g., about aminoacid residues 882-1147 of SEQ ID NO: 11). CARD-10 possesses an SH3domain (e.g., about amino acid residues 704-772 of SEQ ID NO: 8) and aGUK domain (e.g., about amino acid residues 830-1032 of SEQ ID NO: 8),as well as a region bearing homology to PDZ domains (e.g., about aminoacid residues 457-703 of SEQ ID NO: 8).

CARD-10 and CARD-11, like CARD-14, contain both a CARD domain and aMAGUK-homology region. The C-terminal MAGUK-homology regions of CARD-10(about amino acid residues 457-1032 of SEQ ID NO: 8) and CARD-11 (aboutamino acid residues 635-1147 of SEQ ID NO: 11) likely function as sitesof protein-protein interaction that lead to the activation of CARD-10and CARD-11 by upstream signaling proteins. CARD-10 and CARD-11 arelikely membrane-associated via PDZ interactions with a receptor. CARD-10and/or CARD-11 may function to transmit signals from a receptor, e.g.,an antigen receptor such as a T cell receptor or cell surfaceimmunoglobulin, leading to the activation of Bcl-10 and the subsequentactivation of NF-κB. The role of CARD-10 and CARD-11 in Bcl-10 and NF-κBpathways makes CARD-10 and CARD-11 therapeutic targets to blocksignaling mediated via these molecules. For example, CARD-10 and/orCARD-11 may function in antigen receptor signaling in B and T cells byrecruiting Bcl-10 into plasma membrane associated complexes that formfollowing receptor engagement. Thus, compounds that modulate CARD-10and/or CARD-11 activity are expected to modulate Bcl-10 signaling, NF-κBsignaling, and/or lymphocyte activation and proliferation.

Each of CARD-9, CARD-10, and CARD-11 are members of a family ofmolecules (the CARD-9, CARD-10, and CARD-11 family, respectively) havingcertain conserved structural and functional features. The term “family”when referring to the protein and nucleic acid molecules of theinvention is intended to mean two or more proteins or nucleic acidmolecules having a common structural domain and having sufficient aminoacid or nucleotide sequence identity as defined herein. Such familymembers can be naturally occurring and can be from either the same ordifferent species. For example, a family can contain a first protein ofhuman origin and a homologue of that protein of murine origin, as wellas a second, distinct protein of human origin and a murine homologue ofthat protein. Members of a family may also have common functionalcharacteristics.

As used herein, the term “sufficiently identical” refers to a firstamino acid or nucleotide sequence which contains a sufficient or minimumnumber of identical or equivalent (e.g., an amino acid residue which hasa similar side chain) amino acid residues or nucleotides to a secondamino acid or nucleotide sequence such that the first and second aminoacid or nucleotide sequences have a common structural domain and/orcommon functional activity. For example, amino acid or nucleotidesequences which contain a common structural domain having about 65%identity, preferably 75% identity, more preferably 85%, 95%, or 98%identity are defined herein as sufficiently identical.

As used interchangeably herein a “CARD-9, CARD-10, or CARD-11 activity”,“biological activity of CARD-9, CARD-10, or CARD-11” or “functionalactivity of CARD-9, CARD-10, or CARD-11”, refers to an activity exertedby a CARD-9, CARD-10, or CARD-11 protein, polypeptide or nucleic acidmolecule on a CARD-9, CARD-10, or CARD-11 responsive cell as determinedin vivo, or in vitro, according to standard techniques. CARD-9, CARD-10,or CARD-11 may act as a pro-apoptotic protein or an anti-apoptoticprotein (i.e., it might act to decrease or increase apoptosis). ACARD-9, CARD-10, or CARD-11 activity can be a direct activity, such asan association with or an enzymatic activity on a second protein or anindirect activity, such as a cellular signaling activity mediated byinteraction of the CARD-9, CARD-10, or CARD-11 protein with a secondprotein.

In one embodiment, a CARD-9, CARD-10, or CARD-11 activity can include atleast one or more of the following activities: (i) the ability tointeract with proteins in an apoptotic, inflammation, and/or immuneactivation signaling pathway; (ii) the ability to interact with aCARD-9, CARD-10, or CARD-11; (iii) the ability to interact with Bcl-10;(iv) the ability to modulate phosphorylation of Bcl-10; (v) the abilityto modulate the activity of NF-kB; (vi) the ability to modulatelymphocyte activation and or proliferation, e.g., antigenreceptor-induced lymphocyte activation; (vii) the ability to regulateCNS development, e.g., neuronal survival and/or neural tube closure;(viii) the ability to interact with a membrane, e.g., a plasma membrane;(ix) the ability to interact, directly or indirectly, with one or moreproteins having a CARD domain, e.g., a caspase, or an IAP (e.g., IAP-1or IAP-2); (x) the ability to modulate the activity of a caspase, e.g.,caspase-9; (xi) the ability to interact with an intracellular targetprotein; and (xii) the ability to interact with a heat shock protein,e.g., an interaction of the coiled-coil domain of CARD-9, CARD-10, orCARD-11 with a heat shock protein. CARD-9, CARD-10, or CARD-11 nucleicacid and polypeptides as well as modulators of activity of expression ofCARD-9, CARD-10, or CARD-11 might be used to modulate an Apaf-lsignaling pathway. CARD-9, CARD-10, or CARD-11 may modulate the activityof a neurotrophin receptor and thus modulate apoptosis of neuronalcells. Accordingly, CARD-9, CARD-10, or CARD-11 nucleic acids andpolypeptides as well as modulators of CARD-9, CARD-10, or CARD-11activity or expression can be used to modulate apoptosis of neurons(e.g., for treatment of neurological disorders, particularlyneurodegenerative disorders).

Accordingly, another embodiment of the invention features isolatedCARD-9, CARD-10, or CARD-11 proteins and polypeptides having a CARD-9,CARD-10, or CARD-11 activity.

Various aspects of the invention are described in further detail in thefollowing subsections.

I. Isolated Nucleic Acid Molecules

One aspect of the invention pertains to isolated nucleic acid moleculesthat encode CARD-9, CARD-10, or CARD-11 proteins or biologically activeportions thereof, as well as nucleic acid molecules sufficient for useas hybridization probes to identify CARD-9, CARD-10, or CARD-11-encodingnucleic acids (e.g., CARD-9, CARD-10, or CARD-11 mRNA) and fragments foruse as PCR primers for the amplification or mutation of CARD-9, CARD-10,or CARD-11 nucleic acid molecules. As used herein, the term “nucleicacid molecule” is intended to include DNA molecules (e.g., cDNA orgenomic DNA) and RNA molecules (e.g., mRNA) and analogs of the DNA orRNA generated using nucleotide analogs. The nucleic acid molecule can besingle-stranded or double-stranded, but preferably is double-strandedDNA.

An “isolated” nucleic acid molecule is one which is separated from othernucleic acid molecules which are present in the natural source of thenucleic acid. Preferably, an “isolated” nucleic acid is free ofsequences (preferably protein encoding sequences) that which naturallyflank the nucleic acid (i.e., sequences located at the 5′ and 3′ ends ofthe nucleic acid) in the genomic DNA of the organism from which thenucleic acid is derived. For example, in various embodiments, theisolated CARD-9, CARD-10, or CARD-11 nucleic acid molecule can containless than about 5 kb, 4 kb, 3 kb, 2 kb, 1 kb, 0.5 kb or 0.1 kb ofnucleotide sequences which naturally flank the nucleic acid molecule ingenomic DNA of the cell from which the nucleic acid is derived.Moreover, an “isolated” nucleic acid molecule, such as a cDNA molecule,can be substantially free of other cellular material, or culture mediumwhen produced by recombinant techniques, or substantially free ofchemical precursors or other chemicals when chemically synthesized.

A nucleic acid molecule of the present invention, e.g., a nucleic acidmolecule having the nucleotide sequence of SEQ ID NO: 1, SEQ ID NO: 3,SEQ ID NO: 4, SEQ ID NO: 6, SEQ ID NO: 7, SEQ ID NO: 9; SEQ ID NO: 10,SEQ ID NO: 12, or a complement of any of these nucleotide sequences, canbe isolated using standard molecular biology techniques and the sequenceinformation provided herein. Using all or portion of the nucleic acidsequences of SEQ ID NO: 1, SEQ ID NO: 3, SEQ ID NO: 4, SEQ ID NO: 6, SEQID NO: 7, SEQ ID NO: 9, SEQ ID NO: 10, or SEQ ID NO: 12 as ahybridization probe, CARD-9, CARD-10, or CARD-11 nucleic acid moleculescan be isolated using standard hybridization and cloning techniques(e.g., as described in Sambrook et al., eds., Molecular Cloning: ALaboratory Manual. 2nd, ed., Cold Spring Harbor Laboratory, Cold SpringHarbor Laboratory Press, Cold Spring Harbor, N.Y., 1989).

A nucleic acid of the invention can be amplified using cDNA, mRNA orgenomic DNA as a template and appropriate oligonucleotide primersaccording to standard PCR amplification techniques. The nucleic acid soamplified can be cloned into an appropriate vector and characterized byDNA sequence analysis. Furthermore, oligonucleotides corresponding toCARD-9, CARD-10, or CARD-11 nucleotide sequences can be prepared bystandard synthetic techniques, e.g., using an automated DNA synthesizer.

In another embodiment, an isolated nucleic acid molecule of theinvention comprises a nucleic acid molecule which is a complement of thenucleotide sequence shown in SEQ ID NO: 1, SEQ ID NO: 3, SEQ ID NO: 4,SEQ ID NO: 6, SEQ ID NO: 7, SEQ ID NO: 9, SEQ ID NO: 10, SEQ ID NO: 12,or a portion thereof. A nucleic acid molecule which is complementary toa given nucleotide sequence is one which is sufficiently complementaryto the given nucleotide sequence that it can hybridize to the givennucleotide sequence thereby forming a stable duplex.

Moreover, the nucleic acid molecule of the invention can comprise only aportion of a nucleic acid sequence encoding CARD-9, CARD-10, or CARD-11,for example, a fragment which can be used as a probe or primer or afragment encoding a biologically active portion of CARD-9, CARD-10, orCARD-11. The nucleotide sequence determined from the cloning of theCARD-9, CARD-10, or CARD-11 gene allows for the generation of probes andprimers designed for use in identifying and/or cloning CARD-9, CARD-10,or CARD-11 homologues in other cell types, e.g., from other tissues, aswell as CARD-9, CARD-10, or CARD-11 homologues and orthologs from othermammals. The probe/primer typically comprises substantially purifiedoligonucleotide. The oligonucleotide typically comprises a region ofnucleotide sequence that hybridizes under stringent conditions to atleast about 12, preferably about 25, more preferably about 50, 75, 100,125, 150, 175, 200, 250, 300, 350 or 400 consecutive nucleotides of thesense or anti-sense sequence of SEQ ID NO: 1, SEQ ID NO: 3, SEQ ID NO:4, SEQ ID NO: 6, SEQ ID NO: 7, SEQ ID NO: 9, SEQ ID NO:10, SEQ ID NO:12, or of a naturally occurring mutant of one of SEQ ID NO: 1, SEQ IDNO: 3, SEQ ID NO: 4, SEQ ID NO: 6, SEQ ID NO: 7, SEQ ID NO: 9, SEQ IDNO: 10, or SEQ ID NO: 12.

Probes based on the CARD-9, CARD-10, or CARD-11 nucleotide sequence canbe used to detect transcripts or genomic sequences encoding the same orsimilar proteins. The probe comprises a label group attached thereto,e.g., a radioisotope, a fluorescent compound, an enzyme, or an enzymeco-factor. Such probes can be used as a part of a diagnostic test kitfor identifying allelic variants and orthologs of the CARD-9, CARD-10,or CARD-11 proteins of the present invention, identifying cells ortissue which mis-express a CARD-9, CARD-10, or CARD-11 protein, such asby measuring a level of a CARD-9, CARD-10, or CARD-11-encoding nucleicacid in a sample of cells from a subject, e.g., detecting CARD-9,CARD-10, or CARD-11 mRNA levels or determining whether a genomic CARD-9,CARD-10, or CARD-11 gene has been mutated or deleted.

A nucleic acid fragment encoding a “biologically active portion” ofCARD-9, CARD-10, or CARD-11 can be prepared by isolating a portion ofSEQ ID NO: 1, SEQ ID NO: 3, SEQ ID NO: 4, SEQ ID NO: 6, SEQ ID NO: 7,SEQ ID NO: 9, SEQ ID NO: 10, or SEQ ID NO: 12, which encodes apolypeptide having a CARD-9, CARD-10, or CARD-11 biological activity,expressing the encoded portion of CARD-9, CARD-10, or CARD-11 protein(e.g., by recombinant expression in vitro) and assessing the activity ofthe encoded portion of CARD-9, CARD-10, or CARD-11. For example, anucleic acid fragment encoding a biologically active portion of CARD-9,CARD-10, or CARD-11 includes a CARD domain, a coiled-coil domain, a PDZdomain, an SH3 domain, or a GUK domain.

The invention further encompasses nucleic acid molecules that differfrom the nucleotide sequence of SEQ ID NO: 1, SEQ ID NO: 3, SEQ ID NO:4, SEQ ID NO: 6, SEQ ID NO: 7, SEQ ID NO: 9, SEQ ID NO: 10, or SEQ IDNO: 12, due to degeneracy of the genetic code and thus encode the sameCARD-9, CARD-10, or CARD-11 protein as that encoded by the nucleotidesequence shown in SEQ ID NO: 1, SEQ ID NO: 3, SEQ ID NO: 4, SEQ ID NO:6, SEQ ID NO: 7, SEQ ID NO: 9, SEQ ID NO: 10, or SEQ ID NO: 12.

In addition to the CARD-9, CARD-10, or CARD-11 nucleotide sequence shownin SEQ ID NO: 1, SEQ ID NO: 3, SEQ ID NO: 4, SEQ ID NO: 6, SEQ ID NO: 7,SEQ ID NO: 9, SEQ ID NO: 10, or SEQ ID NO: 12, it will be appreciated bythose skilled in the art that DNA sequence polymorphisms that lead tochanges in the amino acid sequences of CARD-9, CARD-10, or CARD-11 mayexist within a population (e.g., the human population). Such geneticpolymorphism in the CARD-9, CARD-10, or CARD-11 gene may exist amongindividuals within a population due to natural allelic variation. Asused herein, the terms “gene” and “recombinant gene” refer to nucleicacid molecules comprising an open reading frame encoding a CARD-9,CARD-10, or CARD-11 protein, preferably a mammalian CARD-9, CARD-10, orCARD-11 protein. Such natural allelic variations can typically result in1-5% variance in the nucleotide sequence of the CARD-9, CARD-10, orCARD-11 gene. Any and all such nucleotide variations and resulting aminoacid polymorphisms in CARD-9, CARD-10, or CARD-11 that are the result ofnatural allelic variation and that do not alter the functional activityof CARD-9, CARD-10, or CARD-11 are intended to be within the scope ofthe invention. Thus, e.g., 1%, 2%, 3%, 4%, or 5% of the amino acids inCARD-9, CARD-10, or CARD-11 (e.g., 1, 2, 3, 4, 5, 6, 8, 10, 15, or 20amino acids) are replaced by another amino acid, preferably byconservative substitution.

Moreover, nucleic acid molecules encoding CARD-9, CARD-10, or CARD-11proteins from other species (CARD-9, CARD-10, or CARD-11orthologs/homologues), which have a nucleotide sequence which differsfrom that of a CARD-9, CARD-10, or CARD-11 disclosed herein, areintended to be within the scope of the invention.

Accordingly, in another embodiment, an isolated nucleic acid molecule ofthe invention is at least 150 (300, 325, 350, 375, 400, 425, 450, 500,550, 600, 650, 700, 800, 900, 1000, 1200, 1400, 1600, 1800, 2000, 2200,2400, 2600, 2800, 3000, 3200, 3400, 3600, 3800, 4000, 4200, or 4250)nucleotides in length and hybridizes under stringent conditions to thenucleic acid molecule comprising the nucleotide sequence, preferably thecoding sequence, of SEQ ID NO: 1, SEQ ID NO: 3, SEQ ID NO: 4, SEQ ID NO:6, SEQ ID NO: 7, SEQ ID NO: 9, SEQ ID NO: 10, or SEQ ID NO: 12.

As used herein, the term “hybridizes under stringent conditions” isintended to describe conditions for hybridization and washing underwhich nucleotide sequences at least 60% (65%, 70%, preferably 75%)identical to each other typically remain hybridized to each other. Suchstringent conditions are known to those skilled in the art and can befound in Current Protocols in Molecular Biology, John Wiley & Sons, N.Y.(1989), 6.3.1-6.3.6. An, non-limiting example of stringent hybridizationconditions are hybridization in 6×sodium chloride/sodium citrate (SSC)at about 45° C., followed by one or more washes in 0.2×SSC, 0.1% SDS at50-65 C (e.g., 50° C. or 60° C. or 65° C.). Preferably, the isolatednucleic acid molecule of the invention that hybridizes under stringentconditions corresponds to a naturally-occurring nucleic acid molecule.As used herein, a “naturally-occurring” nucleic acid molecule refers toan RNA or DNA molecule having a nucleotide sequence that occurs in ahuman cell in nature (e.g., encodes a natural protein).

In addition to naturally-occurring allelic variants of the CARD-9,CARD-10, or CARD-11 sequence that may exist in the population, theskilled artisan will further appreciate that changes can be introducedby mutation into the nucleotide sequence of SEQ ID NO: 1, SEQ ID NO: 3,SEQ ID NO: 4, SEQ ID NO: 6, SEQ ID NO: 7, SEQ ID NO: 9, SEQ ID NO: 10,or SEQ ID NO: 12, thereby leading to changes in the amino acid sequenceof the encoded protein without altering the functional ability of theprotein. For example, one can make nucleotide substitutions leading toamino acid substitutions at “non-essential” amino acid residues. A“non-essential” amino acid residue is a residue that can be altered fromthe wild-type sequence of CARD-9, CARD-10, or CARD-11 protein withoutaltering the biological activity, whereas an “essential” amino acidresidue is required for biological activity. For example, amino acidresidues that are conserved among the CARD-9, CARD-10, or CARD-11,proteins of various species are predicted to be particularly unamenableto alteration.

For example, preferred CARD-9, CARD-10, or CARD-11 proteins of thepresent invention contain at least one CARD domain and at least onecoiled-coil domain. Additionally a CARD-10 and CARD-11 protein alsocontains at least one SH3 domain and at least one GUK domain. A CARD-11protein also contains at least one PDZ domain. Such conserved domainsare less likely to be amenable to mutation. Other amino acid residues,however, (e.g., those that are not conserved or only semi-conservedamong CARD-9, CARD-10, or CARD-11 of various species) may not beessential for activity and thus are likely to be amenable to alteration.

Accordingly, another aspect of the invention pertains to nucleic acidmolecules encoding CARD-9, CARD-10, or CARD-11 proteins that containchanges in amino acid residues that are not essential for activity. SuchCARD-9, CARD-10, or CARD-11 proteins differ in amino acid sequence fromSEQ ID NO: 2, SEQ ID NO: 5, SEQ ID NO: 8, or SEQ ID NO: 11 and yetretain biological activity. In one embodiment, the isolated nucleic acidmolecule includes a nucleotide sequence encoding a protein that includesan amino acid sequence that is at least about 45% identical, 65%, 75%,85%, 95%, or 98% identical to the amino acid sequence of SEQ ID NO: 2,SEQ ID NO: 5, SEQ ID NO: 8, or SEQ ID NO: 11. An isolated nucleic acidmolecule encoding a CARD-9, CARD-10, or CARD-11 protein having asequence which differs from that of SEQ ID NO: 1, SEQ ID NO: 3, SEQ IDNO: 4, SEQ ID NO: 6, SEQ ID NO: 7, SEQ ID NO: 9, SEQ ID NO: 10, or SEQID NO: 12, can be created by introducing one or more nucleotidesubstitutions, additions or deletions into the nucleotide sequence ofCARD-9, CARD-10, or CARD-11 (SEQ ID NO: 1, SEQ ID NO: 3, SEQ ID NO: 4,SEQ ID NO: 6, SEQ ID NO: 7, SEQ ID NO: 9, SEQ ID NO: 10, or SEQ ID NO:12) such that one or more amino acid substitutions, additions ordeletions are introduced into the encoded protein. Mutations can beintroduced by standard techniques, such as site-directed mutagenesis andPCR-mediated mutagenesis. Preferably, conservative amino acidsubstitutions are made at one or more predicted non-essential amino acidresidues. Thus, for example, 1%, 2%, 3%, 5%, or 10% of the amino acidscan be replaced by conservative substitution. A “conservative amino acidsubstitution” is one in which the amino acid residue is replaced with anamino acid residue having a similar side chain. Families of amino acidresidues having similar side chains have been defined in the art. Thesefamilies include amino acids with basic side chains (e.g., lysine,arginine, histidine), acidic side chains (e.g., aspartic acid, glutamicacid), uncharged polar side chains (e.g., glycine, asparagine,glutamine, serine, threonine, tyrosine, cysteine), nonpolar side chains(e.g., alanine, valine, leucine, isoleucine, proline, phenylalanine,methionine, tryptophan), beta-branched side chains (e.g., threonine,valine, isoleucine) and aromatic side chains (e.g., tyrosine,phenylalanine, tryptophan, histidine). Thus, a predicted nonessentialamino acid residue in CARD-9, CARD-10, or CARD-11 is preferably replacedwith another amino acid residue from the same side chain family.Alternatively, mutations can be introduced randomly along all or part ofa CARD-9, CARD-10, or CARD-11 coding sequence, such as by saturationmutagenesis, and the resultant mutants can be screened for CARD-9,CARD-10, or CARD-11 biological activity to identify mutants that retainactivity. Following mutagenesis, the encoded protein can be expressedrecombinantly and the activity of the protein can be determined.

In an embodiment, a mutant CARD-9, CARD-10, or CARD-11 protein can beassayed for: (1) the ability to form protein:protein interactions withproteins in the apoptotic signaling pathway; (2) the ability to bind aCARD-9, CARD-10, or CARD-11 ligand; or (3) the ability to bind to anintracellular target protein.

The present invention encompasses antisense nucleic acid molecules,i.e., molecules which are complementary to a sense nucleic acid encodinga protein, e.g., complementary to the coding strand of a double-strandedcDNA molecule or complementary to an mRNA sequence. Accordingly, anantisense nucleic acid can hydrogen bond to a sense nucleic acid. Theantisense nucleic acid can be complementary to an entire CARD-9,CARD-10, or CARD-11 coding strand, or to only a portion thereof, e.g.,all or part of the protein coding region (or open reading frame). Anantisense nucleic acid molecule can be antisense to a noncoding regionof the coding strand of a nucleotide sequence encoding CARD-9, CARD-10,or CARD-11. The noncoding regions (“5′ and 3′ untranslated regions”) arethe 5′ and 3′ sequences that flank the coding region and are nottranslated into amino acids. Given the coding strand sequences encodingCARD-9, CARD-10, or CARD-11 disclosed herein, antisense nucleic acids ofthe invention can be designed according to the rules of Watson and Crickbase pairing. The antisense nucleic acid molecule can be complementaryto the entire coding region of CARD-9, CARD-10, or CARD-11 mRNA, butmore preferably is an oligonucleotide which is antisense to only aportion of the coding or noncoding region of CARD-9, CARD-10, or CARD-11mRNA. For example, the antisense oligonucleotide can be complementary tothe region surrounding the translation start site of CARD-9, CARD-10, orCARD-11 mRNA. An antisense oligonucleotide can be, for example, about 5,10, 15, 20, 25, 30, 35, 40, 45 or 50 nucleotides in length. An antisensenucleic acid of the invention can be constructed using chemicalsynthesis and enzymatic ligation reactions using procedures known in theart. For example, an antisense nucleic acid (e.g., an antisenseoligonucleotide) can be chemically synthesized using naturally occurringnucleotides or variously modified nucleotides designed to increase thebiological stability of the molecules or to increase the physicalstability of the duplex formed between the antisense and sense nucleicacids, e.g., phosphorothioate derivatives and acridine substitutednucleotides can be used. Examples of modified nucleotides which can beused to generate the antisense nucleic acid include 5-fluorouracil,5-bromouracil, 5-chlorouracil, 5-iodouracil, hypoxanthine, xanthine,4-acetylcytosine, 5-(carboxyhydroxylmethyl) uracil,5-carboxymethylaminomethyl-2-thiouridine,5-carboxymethylaminomethyluracil, dihydrouracil,beta-D-galactosylqueosine, inosine, N6-isopentenyladenine,1-methylguanine, 1-methylinosine, 2,2-dimethylguanine, 2-methyladenine,2-methylguanine, 3-methylcytosine, 5-methylcytosine, N6-adenine,7-methylguanine, 5-methylaminomethyluracil,5-methoxyaminomethyl-2-thiouracil, beta-D-mannosylqueosine,5′-methoxycarboxymethyluracil, 5-methoxyuracil,2-methylthio-N6-isopentenyladenine, uracil-5-oxyacetic acid (v),wybutoxosine, pseudouracil, queosine, 2-thiocytosine,5-methyl-2-thiouracil, 2-thiouracil, 4-thiouracil, 5-methyluracil,uracil-5-oxyacetic acid methylester, uracil-5-oxyacetic acid (v),5-methyl-2-thiouracil, 3-(3-aino-3-N-2-carboxypropyl) uracil, (acp3)w,and 2,6-diaminopurine. Alternatively, the antisense nucleic acid can beproduced biologically using an expression vector into which a nucleicacid has been subcloned in an antisense orientation (i.e., RNAtranscribed from the inserted nucleic acid will be of an antisenseorientation to a target nucleic acid of interest, described further inthe following subsection).

The antisense nucleic acid molecules of the invention are typicallyadministered to a subject or generated in situ such that they hybridizewith or bind to cellular mRNA and/or genomic DNA encoding a CARD-9,CARD-10, or CARD-11 protein to thereby inhibit expression of theprotein, e.g., by inhibiting transcription and/or translation. Thehybridization can be by conventional nucleotide complementarity to forma stable duplex, or, for example, in the case of an antisense nucleicacid molecule which binds to DNA duplexes, through specific interactionsin the major groove of the double helix. An antisense nucleic acidmolecule of the invention can be administered by direct injection at atissue site. Alternatively, antisense nucleic acid molecules can bemodified to target selected cells and then administered systemically.For example, for systemic administration, antisense molecules can bemodified such that they specifically bind to receptors or antigensexpressed on a selected cell surface, e.g., by linking the antisensenucleic acid molecules to peptides or antibodies which bind to cellsurface receptors or antigens. The antisense nucleic acid molecules canalso be delivered to cells using the vectors described herein. Toachieve sufficient intracellular concentrations of the antisensemolecules, vector constructs in which the antisense nucleic acidmolecule is placed under the control of a strong pol II or pol IIIpromoter are preferred.

An antisense nucleic acid molecule of the invention can be an a-anomericnucleic acid molecule. An α-anomeric nucleic acid molecule formsspecific double-stranded hybrids with complementary RNA in which,contrary to the usual β-units, the strands run parallel to each other(Gaultier et al. (1987) Nucleic Acids. Res. 15:6625-6641). The antisensenucleic acid molecule can also comprise a 2′-o-methylribonucleotide(Inoue et al. (1987) Nucleic Acids Res. 15:6131-6148) or a chimericRNA-DNA analogue (Inoue et al. (1987) FEBS Lett. 215:327-330).

The invention also encompasses ribozymes. Ribozymes are catalytic RNAmolecules with ribonuclease activity which are capable of cleaving asingle-stranded nucleic acid, such as an mRNA, to which they have acomplementary region. Thus, ribozymes (e.g., hammerhead ribozymes(described in Haselhoff and Gerlach (1988) Nature 334:585-591)) can beused to catalytically cleave CARD-9, CARD-10, or CARD-11 mRNAtranscripts to thereby inhibit translation of CARD-9, CARD-10, orCARD-11 mRNA. A ribozyme having specificity for a CARD-9, CARD-10, orCARD-11-encoding nucleic acid can be designed based upon the nucleotidesequence of a CARD-9, CARD-10, or CARD-11 cDNA disclosed herein. Forexample, a derivative of a Tetrahymena L-19 IVS RNA can be constructedin which the nucleotide sequence of the active site is complementary tothe nucleotide sequence to be cleaved in a CARD-9, CARD-10, orCARD-11-encoding mRNA. See, e.g., Cech et al. U.S. Pat. No. 4,987,071;and Cech et al. U.S. Pat. No. 5,116,742. Alternatively, CARD-9, CARD-10,or CARD-11 mRNA can be used to select a catalytic RNA having a specificribonuclease activity from a pool of RNA molecules. See, e.g., Barteland Szostak (1993) Science 261:1411-1418.

The invention also encompasses nucleic acid molecules which form triplehelical structures. For example, CARD-9, CARD-10, or CARD-11 geneexpression can be inhibited by targeting nucleotide sequencescomplementary to the regulatory region of the CARD-9, CARD-10, orCARD-11 (e.g., the CARD-9, CARD-10, or CARD-11 promoter and/orenhancers) to form triple helical structures that prevent transcriptionof the CARD-9, CARD-10, or CARD-11 gene in target cells. See generally,Helene (1991) Anticancer Drug Des. 6(6):569-84; Helene (1992) Ann. N.Y.Acad. Sci. 660:27-36; and Maher (1992) Bioassays 14(12):807-15.

In embodiments, the nucleic acid molecules of the invention can bemodified at the base moiety, sugar moiety or phosphate backbone toimprove, e.g., the stability, hybridization, or solubility of themolecule. For example, the deoxyribose phosphate backbone of the nucleicacids can be modified to generate peptide nucleic acids (see Hyrup etal. (1996) Bioorganic & Medicinal Chemistry 4(1):5-23). As used herein,the terms “peptide nucleic acids” or “PNAs” refer to nucleic acidmimics, e.g., DNA mimics, in which the deoxyribose phosphate backbone isreplaced by a pseudopeptide backbone and only the four naturalnucleobases are retained. The neutral backbone of PNAs has been shown toallow for specific hybridization to DNA and RNA under conditions of lowionic strength. The synthesis of PNA oligomers can be performed usingstandard solid phase peptide synthesis protocols as described in Hyrupet al. (1996) supra; Perry-O'Keefe et al. (1996) Proc. Natl. Acad. Sci.USA 93:14670-675.

PNAs of CARD-9, CARD-10, or CARD-11 can be used for therapeutic anddiagnostic applications. For example, PNAs can be used as antisense orantigene agents for sequence-specific modulation of gene expression by,e.g., inducing transcription or translation arrest or inhibitingreplication. PNAs of CARD-9, CARD-10, or CARD-11 can also be used, e.g.,in the analysis of single base pair mutations in a gene by, e.g., PNAdirected PCR clamping; as artificial restriction enzymes when used incombination with other enzymes, e.g., S1 nucleases (Hyrup (1996) supra;or as probes or primers for DNA sequence and hybridization (Hyrup (1996)supra; Perry-O'Keefe et al. (1996) Proc. Natl. Acad. Sci. USA 93:14670-675).

In another embodiment, PNAs of CARD-9, CARD-10, or CARD-11 can bemodified, e.g., to enhance their stability or cellular uptake, byattaching lipophilic or other helper groups to PNA, by the formation ofPNA-DNA chimeras, or by the use of liposomes or other techniques of drugdelivery known in the art. For example, PNA-DNA chimeras of CARD-9,CARD-10, or CARD-11 can be generated which may combine the advantageousproperties of PNA and DNA. Such chimeras allow DNA recognition enzymes,e.g., RNAse H and DNA polymerases, to interact with the DNA portionwhile the PNA portion would provide high binding affinity andspecificity. PNA-DNA chimeras can be linked using linkers of appropriatelengths selected in terms of base stacking, number of bonds between thenucleobases, and orientation (Hyrup (1996) supra). The synthesis ofPNA-DNA chimeras can be preformed as described in Hyrup (1996) supra andFinn et al. (1996) Nucleic Acids Research 24(17):3357-63. For example, aDNA chain can be synthesized on a solid support using standardphosphoramidite coupling chemistry and modified nucleoside analogs,e.g., 5′-(4-methoxytrityl)amino-5′-deoxy-thymidine phosphoramidite, canbe used as a between the PNA and the 5′ end of DNA (Mag et al. (1989)Nucleic Acid Res. 17:5973-88). PNA monomers are then coupled in astepwise manner to produce a chimeric molecule with a 5′ PNA segment anda 3′ DNA segment (Finn et al. (1996) Nucleic Acids Research24(17):3357-63). Alternatively, chimeric molecules can be synthesizedwith a 5′ DNA segment and a 3′ PNA segment (Peterser et al. (1975)Bioorganic Med. Chem. Lett. 5:1119-11124).

In other embodiments, the oligonucleotide may include other appendedgroups such as peptides (e.g., for targeting host cell receptors invivo), or agents facilitating transport across the cell membrane (see,e.g., Letsinger et al. (1989) Proc. Natl. Acad. Sci. USA 86:6553-6556;Lemaitre et al. (1987) Proc. Natl. Acad. Sci. USA 84:648-652; PCTPublication No. WO 88/09810) or the blood-brain barrier (see, e.g., PCTPublication No. W0 89/10134). In addition, oligonucleotides can bemodified with hybridization-triggered cleavage agents (see, e.g., Krolet al. (1988) Bio/Techniques 6:958-976) or intercalating agents (see,e.g., Zon (1988) Pharm. Res. 5:539-549). To this end, theoligonucleotide may be conjugated to another molecule, e.g., a peptide,hybridization triggered cross-linking agent, transport agent,hybridization-triggered cleavage agent, etc.

II. Isolated CARD-9, CARD-10, or CARD-11 Proteins and Anti-CARD-9,CARD-10, or CARD-11 Antibodies

One aspect of the invention pertains to isolated CARD-9, CARD-10, orCARD-11 proteins, and biologically active portions thereof, as well aspolypeptide fragments suitable for use as immunogens to raiseanti-CARD-9, CARD-10, or CARD-11 antibodies. In one embodiment, nativeCARD-9, CARD-10, or CARD-11 proteins can be isolated from cells ortissue sources by an appropriate purification scheme using standardprotein purification techniques. In another embodiment, CARD-9, CARD-10,or CARD-11 proteins are produced by recombinant DNA techniques.Alternative to recombinant expression, a CARD-9, CARD-10, or CARD-11protein or polypeptide can be synthesized chemically using standardpeptide synthesis techniques.

An “isolated” or “purified” protein or biologically active portionthereof is substantially free of cellular material or othercontaminating proteins from the cell or tissue source from which theCARD-9, CARD-10, or CARD-11 protein is derived, or substantially freefrom chemical precursors or other chemicals when chemically synthesized.The language “substantially free of cellular material” includespreparations of CARD-9, CARD-10, or CARD-11 protein in which the proteinis separated from cellular components of the cells from which it isisolated or recombinantly produced. Thus, CARD-9, CARD-10, or CARD-11protein that is substantially free of cellular material includespreparations of CARD-9, CARD-10, or CARD-11 protein having less thanabout 30%, 20%, 10%, or 5% (by dry weight) of non-CARD-9, CARD-10, orCARD-11 protein (also referred to herein as a “contaminating protein”).When the CARD-9, CARD-10, or CARD-11 protein or biologically activeportion thereof is recombinantly produced, it is also preferablysubstantially free of culture medium, i.e., culture medium representsless than about 20%, 10%, or 5% of the volume of the proteinpreparation. When CARD-9, CARD-10, or CARD-11 protein is produced bychemical synthesis, it is preferably substantially free of chemicalprecursors or other chemicals, i.e., it is separated from chemicalprecursors or other chemicals which are involved in the synthesis of theprotein. Accordingly such preparations of CARD-9, CARD-10, or CARD-11protein have less than about 30%, 20%, 10%, 5% (by dry weight) ofchemical precursors or non-CARD-9, CARD-10, or CARD-11 chemicals.

Biologically active portions of a CARD-9, CARD-10, or CARD-11 proteininclude peptides comprising amino acid sequences sufficiently identicalto or derived from the amino acid sequence of the CARD-9, CARD-10, orCARD-11 protein (e.g., the amino acid sequence shown in SEQ ID NO: 2,SEQ ID NO: 5, SEQ ID NO: 8, or SEQ ID NO: 11), which include less aminoacids than the full length CARD-9, CARD-10, or CARD-11 protein, andexhibit at least one activity of a CARD-9, CARD-10, or CARD-11 protein.Typically, biologically active portions comprise a domain or motif withat least one activity of the CARD-9, CARD-10, or CARD-11 protein. Abiologically active portion of a CARD-9, CARD-10, or CARD-11 protein canbe a polypeptide which is, for example, 10, 25, 50, 100, 150, 200, 250,300 or more amino acids in length. Preferred biologically activepolypeptides include one or more identified CARD-9, CARD-10, or CARD-11structural domains, e.g., the CARD domain, the coiled-coil domain, thePDZ domain, the SH3 domain, or the GUK domain.

Moreover, other biologically active portions, in which other regions ofthe protein are deleted, can be prepared by recombinant techniques andevaluated for one or more of the functional activities of a nativeCARD-9, CARD-10, or CARD-11 protein.

Rat CARD-9, human CARD-9, human CARD-10, and human CARD-11 protein havethe amino acid sequences of SEQ ID NO: 2, SEQ ID NO: 5, SEQ ID NO: 8, orSEQ ID NO: 11. Other useful CARD-9, CARD-10, or CARD-11 proteins aresubstantially identical to SEQ ID NO: 2, SEQ ID NO: 5, SEQ ID NO: 8, orSEQ ID NO: 11 and retain the functional activity of the protein of SEQID NO: 2, SEQ ID NO: 5, SEQ ID NO: 8, or SEQ ID NO: 11, yet differ inamino acid sequence due to natural allelic variation or mutagenesis.

A useful CARD-9, CARD-10, or CARD-11 protein is a protein which includesan amino acid sequence at least about 45%, preferably 55%, 65%, 75%,85%, 95%, or 99% identical to the amino acid sequence of SEQ ID NO: 2,SEQ ID NO: 5, SEQ ID NO: 8, or SEQ ID NO: 11, and retains the functionalactivity of the CARD-9, CARD-10, or CARD-11 protein of SEQ ID NO: 2, SEQID NO: 5, SEQ ID NO: 8, or SEQ ID NO: 11.

The determination of percent homology between two sequences can beaccomplished using a mathematical algorithm. A preferred, non-limitingexample of a mathematical algorithm utilized for the comparison of twosequences is the algorithm of Karlin and Altschul (1990) Proc. Nat'lAcad. Sci. USA 87:2264-2268, modified as in Karlin and Altschul (1993)Proc. Nat'l Acad. Sci. USA 90:5873-5877. Such an algorithm isincorporated into the NBLAST and XBLAST programs of Altschul, et al.(1990) J. Mol. Biol. 215:403-410. BLAST nucleotide searches can beperformed with the NBLAST program, score=100, wordlength=12 to obtainnucleotide sequences similar or homologous to nucleic acid molecules ofthe invention. To obtain gapped alignments for comparison purposes,Gapped BLAST can be utilized as described in Altschul et al. (1997)Nucleic Acids Res. 25:3389-3402. When utilizing BLAST and Gapped BLASTprograms, the default parameters of the respective programs (e.g.,XBLAST and NBLAST) can be used. See the website for National Center forBiotechnology Information,Bethesda, MD. Another preferred, non-limitingexample of a mathematical algorithm utilized for the comparison ofsequences is the algorithm of Myers and Miller, CABIOS (1989). Such analgorithm is incorporated into the ALIGN program (version 2.0) which ispart of the GCG sequence alignment software package. When utilizing theALIGN program for comparing amino acid sequences, a PAM120 weightresidue table, a gap length penalty of 12, and a gap penalty of 4 can beused. When utilizing the ALIGN program for comparing nucleic acidsequences, a gap length penalty of 12, and a gap penalty of 4 can beused.

Another preferred example of a mathematical algorithm utilized for thecomparison of sequences is the Needleman and Wunsch (J. Mol. Biol.(1970) 48:444-453) algorithm which has been incorporated into the GAPprogram in the GCG software package (available at the bioinformaticspage of the website maintained by Accelrys, Inc.,San Diego, Calif.,USA), using either a Blossom 62 matrix or a PAM250 matrix, and a gapweight of 16, 14, 12, 10, 8, 6, or 4 and a length weight of 1, 2, 3, 4,5, or 6. In yet another preferred embodiment, the percent identitybetween two nucleotide sequences is determined using the GAP program inthe GCG software package, using a NWSgapdna.CMP matrix and a gap weightof 40, 50, 60, 70, or 80 and a length weight of 1, 2, 3, 4, 5, or 6.

The percent identity between two sequences can be determined usingtechniques similar to those described above, with or without allowinggaps. In calculating percent identity, typically exact matches arecounted.

The invention also provides CARD-9, CARD-10, or CARD-11 chimeric orfusion proteins. As used herein, a CARD-9, CARD-10, or CARD-11 “chimericprotein” or “fusion protein” comprises a CARD-9, CARD-10, or CARD-11polypeptide operatively linked to a non-CARD-9, CARD-10, or CARD-11polypeptide. A “CARD-9, CARD-10, or CARD-11 polypeptide” refers to apolypeptide having an amino acid sequence corresponding to all or aportion (preferably a biologically active portion) of a CARD-9, CARD-10,or CARD-11, whereas a “non-CARD-9, CARD-10, or CARD-11 polypeptide”refers to a polypeptide having an amino acid sequence corresponding to aprotein which is not substantially identical to the CARD-9, CARD-10, orCARD-11 protein, e.g., a protein which is different from the CARD-9,CARD-10, or CARD-11 proteins and which is derived from the same or adifferent organism. Within the fusion protein, the term “operativelylinked” is intended to indicate that the CARD-9, CARD-10, or CARD-11polypeptide and the non-CARD-9, CARD-10, or CARD-11 polypeptide arefused in-frame to each other. The heterologous polypeptide can be fusedto the N-terminus or C-terminus of the CARD-9, CARD-10, or CARD-11polypeptide.

One useful fusion protein is a GST fusion protein in which the CARD-9,CARD-10, or CARD-11 sequences are fused to the C-terminus of the GSTsequences. Such fusion proteins can facilitate the purification ofrecombinant CARD-9, CARD-10, or CARD-11. In another embodiment, thefusion protein contains a signal sequence from another protein. Incertain host cells (e.g., mammalian host cells), expression and/orsecretion of CARD-9, CARD-10, or CARD-11 can be increased through use ofa heterologous signal sequence. For example, the gp67 secretory sequenceof the baculovirus envelope protein can be used as a heterologous signalsequence (Current Protocols in Molecular Biology, Ausubel et al., eds.,John Wiley & Sons, 1992). Other examples of eukaryotic heterologoussignal sequences include the secretory sequences of melittin and humanplacental alkaline phosphatase (Stratagene; La Jolla, Calif.). In yetanother example, useful prokaryotic heterologous signal sequencesinclude the phoA secretory signal (Molecular cloning, Sambrook et al,second edition, Cold spring harbor laboratory press, 1989) and theprotein A secretory signal (Pharmacia Biotech; Piscataway, N.J.).

In yet another embodiment, the fusion protein is a CARD-9, CARD-10, orCARD-11-immunoglobulin fusion protein in which all or part of CARD-9,CARD-10, or CARD-11 is fused to sequences derived from a member of theimmunoglobulin protein family. The CARD-9, CARD-10, orCARD-11-immunoglobulin fusion proteins of the invention can beincorporated into pharmaceutical compositions and administered to asubject to inhibit an interaction between a CARD-9, CARD-10, or CARD-11ligand and a CARD-9, CARD-10, or CARD-11 protein on the surface of acell, to thereby suppress CARD-9, CARD-10, or CARD-11-mediated signaltransduction in vivo. The CARD-9, CARD-10, or CARD-11-immunoglobulinfusion proteins can be used to affect the bioavailability of a CARD-9,CARD-10, or CARD-11 cognate ligand. Inhibition of the CARD-9, CARD-10,or CARD-11 ligand/ CARD-9, CARD-10, or CARD-11 interaction may be usefultherapeutically for both the treatment of proliferative anddifferentiative disorders, as well as modulating (e.g., promoting orinhibiting) cell survival. Moreover, the CARD-9, CARD-10, orCARD-11-immunoglobulin fusion proteins of the invention can be used asimmunogens to produce anti-CARD-9, CARD-10, or CARD-11 antibodies in asubject, to purify CARD-9, CARD-10, or CARD-11 ligands and in screeningassays to identify molecules which inhibit the interaction of CARD-9,CARD-10, or CARD-11 with a CARD-9, CARD-10, or CARD-11 ligand.

Preferably, a CARD-9, CARD-10, or CARD-11 chimeric or fusion protein ofthe invention is produced by standard recombinant DNA techniques. Forexample, DNA fragments coding for the different polypeptide sequencesare ligated together in-frame in accordance with conventionaltechniques, for example by employing blunt-ended or stagger-endedtermini for ligation, restriction enzyme digestion to provide forappropriate termini, filling-in of cohesive ends as appropriate,alkaline phosphatase treatment to avoid undesirable joining, andenzymatic ligation. In another embodiment, the fusion gene can besynthesized by conventional techniques including automated DNAsynthesizers. Alternatively, PCR amplification of gene fragments can becarried out using anchor primers which give rise to complementaryoverhangs between two consecutive gene fragments which can subsequentlybe annealed and reamplified to generate a chimeric gene sequence (see,e.g., Current Protocols in Molecular Biology, Ausubel et al. eds., JohnWiley & Sons: 1992). Moreover, many expression vectors are commerciallyavailable that already encode a fusion moiety (e.g., a GST polypeptide).A CARD-9, CARD-10, or CARD-11-encoding nucleic acid can be cloned intosuch an expression vector such that the fusion moiety is linked in-frameto the CARD-9, CARD-10, or CARD-11 protein.

The present invention also pertains to variants of the CARD-9, CARD-10,or CARD-11 proteins which function as either CARD-9, CARD-10, or CARD-11agonists (mimetics) or as CARD-9, CARD-10, or CARD-11 antagonists.Variants of the CARD-9, CARD-10, or CARD-11 proteins can be generated bymutagenesis, e.g., discrete point mutation or truncation of the CARD-9,CARD-10, or CARD-11 proteins. An agonist of the CARD-9, CARD-10, orCARD-11 protein can retain substantially the same, or a subset, of thebiological activities of the naturally occurring form of the CARD-9,CARD-10, or CARD-11 protein. An antagonist of the CARD-9, CARD-10, orCARD-11 protein can inhibit one or more of the activities of thenaturally occurring form of the CARD-9, CARD-10, or CARD-11 protein by,for example, competitively binding to a downstream or upstream member ofa cellular signaling cascade which includes the CARD-9, CARD-10, orCARD-11 protein. Thus, specific biological effects can be elicited bytreatment with a variant of limited function. Treatment of a subjectwith a variant having a subset of the biological activities of thenaturally occurring form of the protein can have fewer side effects in asubject relative to treatment with the naturally occurring form of theCARD-9, CARD-10, or CARD-11 proteins.

Variants of the CARD-9, CARD-10, or CARD-11 protein which function aseither CARD-9, CARD-10, or CARD-11 agonists (mimetics) or as CARD-9,CARD-10, or CARD-11 antagonists can be identified by screeningcombinatorial libraries of mutants, e.g., truncation mutants of theCARD-9, CARD-10, or CARD-11 protein for CARD-9, CARD-10, or CARD-11protein agonist or antagonist activity. In one embodiment, a variegatedlibrary of CARD-9, CARD-10, or CARD-11 variants is generated bycombinatorial mutagenesis at the nucleic acid level and is encoded by avariegated gene library. A variegated library of CARD-9, CARD-10, orCARD-11 variants can be produced by, for example, enzymatically ligatinga mixture of synthetic oligonucleotides into gene sequences such that adegenerate set of potential CARD-9, CARD-10, or CARD-11 sequences isexpressible as individual polypeptides, or alternatively, as a set oflarger fusion proteins (e.g., for phage display) containing the set ofCARD-9, CARD-10, or CARD-11 sequences therein. There are a variety ofmethods which can be used to produce libraries of potential CARD-9,CARD-10, or CARD-11 variants from a degenerate oligonucleotide sequence.Chemical synthesis of a degenerate gene sequence can be performed in anautomatic DNA synthesizer, and the synthetic gene then ligated into anappropriate expression vector. Use of a degenerate set of genes allowsfor the provision, in one mixture, of all of the sequences encoding thedesired set of potential CARD-9, CARD-10, or CARD-11 sequences. Methodsfor synthesizing degenerate oligonucleotides are known in the art (see,e.g., Narang (1983) Tetrahedron 39:3; Itakura et al. (1984) Annu. Rev.Biochem. 53:323; Itakura et al. (1984) Science 198:1056; Ike et al.(1983) Nucleic Acid Res. 11:477).

Useful fragments of CARD-9, CARD-10, or CARD-11, include fragmentscomprising or consisting of a domain or subdomain described herein,e.g., a CARD domain, a coiled-coil domain, a PDZ domain, an SH3 domain,or a GUK domain.

In addition, libraries of fragments of the CARD-9, CARD-10, or CARD-11protein coding sequence can be used to generate a variegated populationof CARD-9, CARD-10, or CARD-11 fragments for screening and subsequentselection of variants of a CARD-9, CARD-10, or CARD-11 protein. In oneembodiment, a library of coding sequence fragments can be generated bytreating a double stranded PCR fragment of a CARD-9, CARD-10, or CARD-11coding sequence with a nuclease under conditions wherein nicking occursonly about once per molecule, denaturing the double stranded DNA,renaturing the DNA to form double stranded DNA which can includesense/antisense pairs from different nicked products, removing singlestranded portions from reformed duplexes by treatment with S1 nuclease,and ligating the resulting fragment library into an expression vector.By this method, an expression library can be derived which encodesN-terminal and internal fragments of various sizes of the CARD-9,CARD-10, or CARD-11 protein.

Several techniques are known in the art for screening gene products ofcombinatorial libraries made by point mutations or truncation, and forscreening cDNA libraries for gene products having a selected property.Such techniques are adaptable for rapid screening of the gene librariesgenerated by the combinatorial mutagenesis of CARD-9, CARD-10, orCARD-11 proteins. The most widely used techniques, which are amenable tohigh through-put analysis, for screening large gene libraries typicallyinclude cloning the gene library into replicable expression vectors,transforming appropriate cells with the resulting library of vectors,and expressing the combinatorial genes under conditions in whichdetection of a desired activity facilitates isolation of the vectorencoding the gene whose product was detected. Recursive ensemblemutagenesis (REM), a technique which enhances the frequency offunctional mutants in the libraries, can be used in combination with thescreening assays to identify CARD-9, CARD-10, or CARD-11 variants (Arkinand Yourvan (1992) Proc. Natl. Acad. Sci. USA 89:7811-7815; Delgrave etal. (1993) Protein Engineering 6(3):327-331).

An isolated CARD-9, CARD-10, or CARD-11 protein, or a portion orfragment thereof, can be used as an immunogen to generate antibodiesthat bind CARD-9, CARD-10, or CARD-11 using standard techniques forpolyclonal and monoclonal antibody preparation. The full-length CARD-9,CARD-10, or CARD-11 protein can be used or, alternatively, the inventionprovides antigenic peptide fragments of CARD-9, CARD-10, or CARD-11 foruse as immunogens. The antigenic peptide of CARD-9, CARD-10, or CARD-11comprises at least 8 (preferably 10, 15, 20, or 30) amino acid residuesof the amino acid sequence shown in SEQ ID NO: 2, SEQ ID NO: 5, SEQ IDNO: 8, or SEQ ID NO: 11 and encompasses an epitope of CARD-9, CARD-10,or CARD-11 such that an antibody raised against the peptide forms aspecific immune complex with CARD-9, CARD-10, or CARD-11.

Useful antibodies include antibodies which bind to a domain or subdomainof CARD-9, CARD-10, or CARD-11 described herein (e.g., a CARD domain, acoiled-coil domain, a PDZ domain, an SH3 domain, or a GUK domain).

Preferred epitopes encompassed by the antigenic peptide are regions ofCARD-9, CARD-10, or CARD-11 that are located on the surface of theprotein, e.g., hydrophilic regions. Other important criteria include apreference for a terminal sequence, high antigenic index (e.g., aspredicted by Jameson-Wolf algorithm), ease of peptide synthesis (e.g.,avoidance of prolines); and high surface probability (e.g., as predictedby the Emini algorithm; FIGS. 3, 7, 12, and 16).

A CARD-9, CARD-10, or CARD-11 immunogen typically is used to prepareantibodies by immunizing a suitable subject, (e.g., rabbit, goat, mouseor other mammal) with the immunogen. An appropriate immunogenicpreparation can contain, for example, recombinantly expressed CARD-9,CARD-10, or CARD-11 protein or a chemically synthesized CARD-9, CARD-10,or CARD-11 polypeptide. The preparation can further include an adjuvant,such as Freund's complete or incomplete adjuvant, or similarimmunostimulatory agent. Immunization of a suitable subject with animmunogenic CARD-9, CARD-10, or CARD-11 preparation induces a polyclonalanti-CARD-9, CARD-10, or CARD-11 antibody response.

Accordingly, another aspect of the invention pertains to anti-CARD-9,CARD-10, or CARD-11 antibodies. The term “antibody” as used hereinrefers to immunoglobulin molecules and immunologically active portionsof immunoglobulin molecules, i.e., molecules that contain an antigenbinding site which specifically binds an antigen, such as CARD-9,CARD-10, or CARD-11. A molecule which specifically binds to CARD-9,CARD-10, or CARD-11 is a molecule which binds CARD-9, CARD-10, orCARD-11, but does not substantially bind other molecules in a sample,e.g., a biological sample, which naturally contains CARD-9, CARD-10, orCARD-11. Examples of immunologically active portions of immunoglobulinmolecules include F(ab) and F(ab′)2 fragments which can be generated bytreating the antibody with an enzyme such as pepsin. The inventionprovides polyclonal and monoclonal antibodies that bind CARD-9, CARD-10,or CARD-11. The term “monoclonal antibody” or “monoclonal antibodycomposition”, as used herein, refers to a population of antibodymolecules that contain only one species of an antigen binding sitecapable of immunoreacting with a particular epitope of CARD-9, CARD-10,or CARD-11. A monoclonal antibody composition thus typically displays asingle binding affinity for a particular CARD-9, CARD-10, or CARD-11protein with which it immunoreacts.

Polyclonal anti-CARD-9, CARD-10, or CARD-11 antibodies can be preparedas described above by immunizing a suitable subject with a CARD-9,CARD-10, or CARD-11 immunogen. The anti-CARD-9, CARD-10, or CARD-11antibody titer in the immunized subject can be monitored over time bystandard techniques, such as with an enzyme linked immunosorbent assay(ELISA) using immobilized CARD-9, CARD-10, or CARD-11. If desired, theantibody molecules directed against CARD-9, CARD-10, or CARD-11 can beisolated from the mammal (e.g., from the blood) and further purified bywell-known techniques, such as protein A chromatography to obtain theIgG fraction. At an appropriate time after immunization, e.g., when theanti-CARD-9, CARD-10, or CARD-11 antibody titers are highest,antibody-producing cells can be obtained from the subject and used toprepare monoclonal antibodies by standard techniques, such as thehybridoma technique originally described by Kohler and Milstein (1975)Nature 256:495-497, the human B cell hybridoma technique (Kozbor et al.(1983) Immunol Today 4:72), the EBV-hybridoma technique (Cole et al.(1985), Monoclonal Antibodies and Cancer Therapy, Alan R. Liss, Inc.,pp. 77-96) or trioma techniques. The technology for producing variousantibodies monoclonal antibody hybridomas is well known (see generallyCurrent Protocols in Immunology (1994) Coligan et al. (eds.) John Wiley& Sons, Inc., New York, N.Y.). Briefly, an immortal cell line (typicallya myeloma) is fused to lymphocytes (typically splenocytes) from a mammalimmunized with a CARD-9, CARD-10, or CARD-11 immunogen as describedabove, and the culture supernatants of the resulting hybridoma cells arescreened to identify a hybridoma producing a monoclonal antibody thatbinds CARD-9, CARD-10, or CARD-11.

Any of the many well known protocols used for fusing lymphocytes andimmortalized cell lines can be applied for the purpose of generating ananti-CARD-9, CARD-10, or CARD-11 monoclonal antibody (see, e.g., CurrentProtocols in Immunology, supra; Galfre et al. (1977) Nature 266:55052;R. H. Kenneth, in Monoclonal Antibodies: A New Dimension In BiologicalAnalyses, Plenum Publishing Corp., New York, N.Y. (1980); and Lerner(1981) Yale J. Biol. Med., 54:387-402). Moreover, the ordinarily skilledworker will appreciate that there are many variations of such methodswhich also would be useful. Typically, the immortal cell line (e.g., amyeloma cell line) is derived from the same mammalian species as thelymphocytes. For example, murine hybridomas can be made by fusinglymphocytes from a mouse immunized with an immunogenic preparation ofthe present invention with an immortalized mouse cell line, e.g., amyeloma cell line that is sensitive to culture medium containinghypoxanthine, aminopterin and thymidine (“HAT medium”). Any of a numberof myeloma cell lines can be used as a fusion partner according tostandard techniques, e.g., the P3-NS1/1-Ag4-1, P3-x63-Ag8.653 orSp2/O-Ag14 myeloma lines. These myeloma lines are available from ATCC.Typically, HAT-sensitive mouse myeloma cells are fused to mousesplenocytes using polyethylene glycol (“PEG”). Hybridoma cells resultingfrom the fusion are then selected using HAT medium, which kills unfusedand unproductively fused myeloma cells (unfused splenocytes die afterseveral days because they are not transformed). Hybridoma cellsproducing a monoclonal antibody of the invention are detected byscreening the hybridoma culture supernatants for antibodies that bindCARD-9, CARD-10, or CARD-11, e.g., using a standard ELISA assay.

Alternative to preparing monoclonal antibody-secreting hybridomas, amonoclonal anti-CARD-9, CARD-10, or CARD-11 antibody can be identifiedand isolated by screening a recombinant combinatorial immunoglobulinlibrary (e.g., an antibody phage display library) with CARD-9, CARD-10,or CARD-11 to thereby isolate immunoglobulin library members that bindCARD-9, CARD-10, or CARD-11. Kits for generating and screening phagedisplay libraries are commercially available (e.g., the PharmaciaRecombinant Phage Antibody System, Catalog No. 27-9400-01; and theStratagene SurfZAP Phage Display Kit, Catalog No. 240612). Additionally,examples of methods and reagents particularly amenable for use ingenerating and screening antibody display library can be found in, forexample, U.S. Pat. No. 5,223,409; PCT Publication No. WO 92/18619; PCTPublication No. WO 91/17271; PCT Publication No. WO 92/20791; PCTPublication No. WO 92/15679; PCT Publication No. WO 93/01288; PCTPublication No. WO 92/01047; PCT Publication No. WO 92/09690; PCTPublication No. WO 90/02809; Fuchs et al. (1991) Bio/Technology9:1370-1372; Hay et al. (1992) Hum. Antibod. Hybridomas 3:81-85; Huse etal. (1989) Science 246:1275-1281; Griffiths et al. (1993) EMBO J.12:725-734.

Additionally, recombinant anti-CARD-9, CARD-10, or CARD-11 antibodies,such as chimeric and humanized monoclonal antibodies, comprising bothhuman and non-human portions, which can be made using standardrecombinant DNA techniques, are within the scope of the invention. Suchchimeric and humanized monoclonal antibodies can be produced byrecombinant DNA techniques known in the art, for example using methodsdescribed in PCT Publication No. WO 87/02671; European PatentApplication 184,187; European Patent Application 171,496; EuropeanPatent Application 173,494; PCT Publication No. WO 86/01533; U.S. Pat.No. 4,816,567; European Patent Application 125,023; Better et al. (1988)Science 240:1041-1043; Liu et al. (1987) Proc. Natl. Acad. Sci. USA84:3439-3443; Liu et al. (1987) J. Immunol. 139:3521-3526; Sun et al.(1987) Proc. Natl. Acad. Sci. USA 84:214-218; Nishimura et al. (1987)Canc. Res. 47:999-1005; Wood et al. (1985) Nature 314:446-449; and Shawet al. (1988) J. Natl. Cancer Inst. 80:1553-1559); Morrison, (1985)Science 229:1202-1207; Oi et al. (1986) Bio/Techniques 4:214; U.S. Pat.No. 5,225,539; Jones et al. (1986) Nature 321:552-525; Verhoeyan et al.(1988) Science 239:1534; and Beidler et al. (1988) J. Immunol.141:4053-4060.

An anti-CARD-9, CARD-10, or CARD-11 antibody (e.g., monoclonal antibody)can be used to isolate CARD-9, CARD-10, or CARD-11 by standardtechniques, such as affinity chromatography or immunoprecipitation. Ananti-CARD-9, CARD-10, or CARD-11 antibody can facilitate thepurification of natural CARD-9, CARD-10, or CARD-11 from cells and ofrecombinantly produced CARD-9, CARD-10, or CARD-11 expressed in hostcells. Moreover, an anti-CARD-9, CARD-10, or CARD-11 antibody can beused to detect CARD-9, CARD-10, or CARD-11 protein (e.g., in a cellularlysate or cell supernatant) in order to evaluate the abundance andpattern of expression of the CARD-9, CARD-10, or CARD-11 protein.Anti-CARD-9, CARD-10, or CARD-11 antibodies can be used diagnosticallyto monitor protein levels in tissue as part of a clinical testingprocedure, e.g., to, for example, determine the efficacy of a giventreatment regimen. Detection can be facilitated by coupling the antibodyto a detectable substance. Examples of detectable substances includevarious enzymes, prosthetic groups, fluorescent materials, luminescentmaterials, bioluminescent materials, and radioactive materials. Examplesof suitable enzymes include horseradish peroxidase, alkalinephosphatase, β-galactosidase, or acetylcholinesterase; examples ofsuitable prosthetic group complexes include streptavidin/biotin andavidin/biotin; examples of suitable fluorescent materials includeumbelliferone, fluorescein, fluorescein isothiocyanate, rhodamine,dichlorotriazinylamine fluorescein, dansyl chloride or phycoerythrin; anexample of a luminescent material includes luminol; examples ofbioluminescent materials include luciferase, luciferin, and aequorin,and examples of suitable radioactive material include ¹²⁵I, ¹³¹I, ³⁵S or³H.

Further, an antibody (or fragment thereof) may be conjugated to atherapeutic moiety such as a cytotoxin, a therapeutic agent or aradioactive metal ion. A cytotoxin or cytotoxic agent includes any agentthat is detrimental to cells. Examples include taxol, cytochalasin B,gramicidin D, ethidium bromide, emetine, mitomycin, etoposide,tenoposide, vincristine, vinblastine, colchicin, doxorubicin,daunorubicin, dihydroxy anthracin dione, mitoxantrone, mithramycin,actinomycin D, 1-dehydrotestosterone, glucocorticoids, procaine,tetracaine, lidocaine, propranolol, and puromycin and analogs orhomologs thereof. Therapeutic agents include, but are not limited to,antimetabolites (e.g., methotrexate, 6-mercaptopurine, 6-thioguanine,cytarabine, 5-fluorouracil decarbazine), alkylating agents (e.g.,mechlorethamine, thioepa chlorambucil, melphalan, carmustine (BSNU) andlomustine (CCNU), cyclothosphamide, busulfan, dibromomannitol,streptozotocin, mitomycin C, and cis-dichlorodiamine platinum (II) (DDP)cisplatin), anthracyclines (e.g., daunorubicin (formerly daunomycin) anddoxorubicin), antibiotics (e.g., dactinomycin (formerly actinomycin),bleomycin, mithramycin, and anthramycin (AMC)), and anti-mitotic agents(e.g., vincristine and vinblastine).

The conjugates of the invention can be used for modifying a givenbiological response. The drug moiety is not to be construed as limitedto classical chemical therapeutic agents. For example, the drug moietymay be a protein or polypeptide possessing a desired biologicalactivity. Such proteins may include, for example, a toxin such as abrin,ricin A, pseudomonas exotoxin, or diphtheria toxin; a protein such astumor necrosis factor, α-interferon, β-interferon, nerve growth factor,platelet derived growth factor, tissue plasminogen activator; or,biological response modifiers such as, for example, lymphokines,interleukin-1 (IL-1), interleukin-2 (IL-2), interleukin-6 (IL-6),granulocyte macrophase colony stimulating factor (GM-CSF), granulocytecolony stimulating factor (G-CSF), or other growth factors.

Techniques for conjugating such therapeutic moiety to antibodies arewell known, see, e.g., Arnon et al., “Monoclonal Antibodies forImmunotargeting of Drugs in Cancer Therapy”, in Monoclonal Antibodiesand Cancer Therapy, Reisfeld et al. (eds.), pp. 243-56 (Alan R. Liss,Inc. 1985); Hellstrom et al., “Antibodies for Drug Delivery”, inControlled Drug Delivery (2nd Ed.), Robinson et al. (eds.), pp. 623-53(Marcel Dekker, Inc. 1987); Thorpe, “Antibody Carriers of CytotoxicAgents in Cancer Therapy: A Review”, in Monoclonal Antibodies '84:Biological and Clinical Applications, Pinchera et al. (eds.), pp.475-506 (1985); “Analysis, Results, and Future Prospective of TheTherapeutic Use of Radiolabeled Antibody In Cancer Therapy”, inMonoclonal Antibodies for Cancer Detection and Therapy, Baldwin et al.(eds.), pp. 303-16 (Academic Press 1985), and Thorpe et al., “ThePreparation and Cytotoxic Properties of Antibody-Toxin Conjugates”,Immunol. Rev., 62:119-58 (1982). Alternatively, an antibody can beconjugated to a second antibody to form an antibody heteroconjugate asdescribed by Segal in U.S. Pat. No. 4,676,980.

In addition, antibodies of the invention, either conjugated or notconjugated to a therapeutic moiety, can be administered together or incombination with a therapeutic moiety such as a cytotoxin, a therapeuticagent or a radioactive metal ion. The order of administration of theantibody and therapeutic moiety can vary. For example, in someembodiments, the antibody is administered concurrently (through the sameor different delivery devices, e.g., syringes) with the therapeuticmoiety. Alternatively, the antibody can be administered separately andprior to the therapeutic moiety. Still alternatively, the therapeuticmoiety is administered separately and prior to the antibody. In manyembodiments, these administration regimens will be continued for days,months or years.

III. Computer Readable Means

The nucleotide or amino acid sequences of the invention are alsoprovided in a variety of mediums to facilitate use thereof. As usedherein, “provided” refers to a manufacture, other than an isolatednucleic acid or amino acid molecule, which contains a nucleotide oramino acid sequence of the present invention. Such a manufactureprovides the nucleotide or amino acid sequences, or a subset thereof(e.g., a subset of open reading frames (ORFs)) in a form which allows askilled artisan to examine the manufacture using means not directlyapplicable to examining the nucleotide or amino acid sequences, or asubset thereof, as they exist in nature or in purified form.

In one application of this embodiment, a nucleotide or amino acidsequence of the present invention can be recorded on computer readablemedia. As used herein, “computer readable media” refers to any mediumthat can be read and accessed directly by a computer. Such mediainclude, but are not limited to: magnetic storage media, such as floppydiscs, hard disc storage medium, and magnetic tape; optical storagemedia such as CD-ROM; electrical storage media such as RAM and ROM; andhybrids of these categories such as magnetic/optical storage media. Thisskilled artisan will readily appreciate how any of the presently knowncomputer readable mediums can be used to create a manufacture comprisingcomputer readable medium having recorded thereon a nucleotide or aminoacid sequence of the present invention.

As used herein, “recorded” refers to a process for storing informationon computer readable medium. The skilled artisan can readily adopt anyof the presently known methods for recording information on computerreadable medium to generate manufactures comprising the nucleotide oramino acid sequence information of the present invention.

A variety of data storage structures are available to a skilled artisanfor creating a computer readable medium having recorded thereon anucleotide or amino acid sequence of the present invention. The choiceof the data storage structure will generally be based on the meanschosen to access the stored information. In addition, a variety of dataprocessor programs and formats can be used to store the nucleotidesequence information of the present invention on computer readablemedium. The sequence information can be represented in a work processingtest file, formatted in commercially-available software such asWordPerfect and Microsoft Word, or represented in the form of an ASCIIfile, stored in a database application, such as DB2, Sybase, Oracle, orthe like. The skilled artisan can readily adapt any number of dataprocessor structuring formats (e.g., text file or database) in order toobtain computer readable medium having recorded thereon the nucleotidesequence information of the present invention.

By providing the nucleotide or amino acid sequences of the invention incomputer readable form, the skilled artisan can routinely access thesequence information for a variety of purposes. For example, one skilledin the art can use the nucleotide or amino acid sequences of theinvention in computer readable form to compare a target sequence or atarget structural motif with the sequence information stored within thedata storage means. Search means are used to identify fragments orregions of the sequences of the invention which match a particulartarget sequence or target motif.

As used herein, a “target sequence” can be any DNA or amino acidsequence of six or more nucleotides or two or more amino acids. Askilled artisan can readily recognize that the longer a target sequenceis, the less likely a target sequence will be present as a randomoccurrence in the database. The most preferred sequence length of atarget sequence is from about 10 to 100 amino acids or from about 30 to300 nucleotide residues. However, it is well recognized thatcommercially important fragments, such as sequence fragments involved ingene expression and protein processing, may be of shorter length.

As used herein, “a target structural motif,” or “target motif,” refersto any rationally selected sequence or combination of sequences in whichthe sequence(s) are chosen based on a three-dimensional configurationwhich is formed upon the folding of the target motif. There are avariety of target motifs know in the art. Protein target motifs include,but are not limited to, enzyme active sites and signal sequences.Nucleic acid target motifs include, but are not limited to, promotersequences, hairpin structures and inducible expression elements (proteinbinding sequences).

Computer software is publicly available which allows a skilled artisanto access sequence information provided in a computer readable mediumfor analysis and comparison to other sequences. A variety of knowalgorithms are disclosed publicly and a variety of commerciallyavailable software for conducting search means are and can be used inthe computer-based systems of the present invention. Examples of suchsoftware includes, but is not limited to, MacPattern (EMBL), BLASTIN andBLASTX (NCBIA).

For example, software which implements the BLAST (Altschul et al. (1990)J. of Mol. Biol. 215:403-410) and BLAZE (Brutlag et al. (1993) Comp.Chem. 17:203-207) search algorithms on a Sybase system can be used toidentify open reading frames (ORFs) of the sequences of the inventionwhich contain homology to ORFs or proteins from other libraries. SuchORFs are protein-encoding fragments and are useful in producingcommercially important proteins such as enzymes used in variousreactions and in the production of commercially useful metabolites.

IV. Recombinant Expression Vectors and Host Cells

Another aspect of the invention pertains to vectors, preferablyexpression vectors, containing a nucleic acid encoding CARD-9, CARD-10,or CARD-11 (or a portion thereof). As used herein, the term “vector”refers to a nucleic acid molecule capable of transporting anothernucleic acid to which it has been linked. One type of vector is a“plasmid”, which refers to a circular double stranded DNA loop intowhich additional DNA segments can be ligated. Another type of vector isa viral vector, wherein additional DNA segments can be ligated into theviral genome. Certain vectors are capable of autonomous replication in ahost cell into which they are introduced (e.g., bacterial vectors havinga bacterial origin of replication and episomal mammalian vectors). Othervectors (e.g., non-episomal mammalian vectors) are integrated into thegenome of a host cell upon introduction into the host cell, and therebyare replicated along with the host genome. Moreover, certain vectors,expression vectors, are capable of directing the expression of genes towhich they are operatively linked. In general, expression vectors ofutility in recombinant DNA techniques are often in the form of plasmids(vectors). However, the invention is intended to include such otherforms of expression vectors, such as viral vectors (e.g., replicationdefective retroviruses, adenoviruses and adeno-associated viruses),which serve equivalent functions.

The recombinant expression vectors of the invention comprise a nucleicacid of the invention in a form suitable for expression of the nucleicacid in a host cell, which means that the recombinant expression vectorsinclude one or more regulatory sequences, selected on the basis of thehost cells to be used for expression, which is operatively linked to thenucleic acid sequence to be expressed. Within a recombinant expressionvector, “operably linked” is intended to mean that the nucleotidesequence of interest is linked to the regulatory sequence(s) in a mannerwhich allows for expression of the nucleotide sequence (e.g., in an invitro transcription/translation system or in a host cell when the vectoris introduced into the host cell). The term “regulatory sequence” isintended to include promoters, enhancers and other expression controlelements (e.g., polyadenylation signals). Such regulatory sequences aredescribed, for example, in Goeddel; Gene Expression Technology: Methodsin Enzymology 185, Academic Press, San Diego, Calif. (1990). Regulatorysequences include those which direct constitutive expression of anucleotide sequence in many types of host cell and those which directexpression of the nucleotide sequence only in certain host cells (e.g.,tissue-specific regulatory sequences). It will be appreciated by thoseskilled in the art that the design of the expression vector can dependon such factors as the choice of the host cell to be transformed, thelevel of expression of protein desired, etc. The expression vectors ofthe invention can be introduced into host cells to thereby produceproteins or peptides, including fusion proteins or peptides, encoded bynucleic acids as described herein (e.g., CARD-9, CARD-10, or CARD-11proteins, mutant forms of CARD-9, CARD-10, or CARD-11, fusion proteins,etc.).

The recombinant expression vectors of the invention can be designed forexpression of CARD-9, CARD-10, or CARD-11 in prokaryotic or eukaryoticcells, e.g., bacterial cells such as E. coli, insect cells (usingbaculovirus expression vectors) yeast cells or mammalian cells. Suitablehost cells are discussed further in Goeddel, Gene Expression Technology:Methods in Enzymology 185, Academic Press, San Diego, Calif. (1990).Alternatively, the recombinant expression vector can be transcribed andtranslated in vitro, for example using T7 promoter regulatory sequencesand T7 polymerase.

Expression of proteins in prokaryotes is most often carried out in E.coli with vectors containing constitutive or inducible promotersdirecting the expression of either fusion or non-fusion proteins. Fusionvectors add a number of amino acids to a protein encoded therein,usually to the amino terminus of the recombinant protein. Such fusionvectors typically serve three purposes: 1) to increase expression ofrecombinant protein; 2) to increase the solubility of the recombinantprotein; and 3) to aid in the purification of the recombinant protein byacting as a ligand in affinity purification. Often, in fusion expressionvectors, a proteolytic cleavage site is introduced at the junction ofthe fusion moiety and the recombinant protein to enable separation ofthe recombinant protein from the fusion moiety subsequent topurification of the fusion protein. Such enzymes, and their cognaterecognition sequences, include Factor Xa, thrombin and enterokinase.Typical fusion expression vectors include pGEX (Pharmacia Biotech Inc;Smith and Johnson (1988) Gene 67:31-40), pMAL (New England Biolabs,Beverly, Mass.) and pRIT5 (Pharmacia, Piscataway, N.J.) which fuseglutathione S-transferase (GST), maltose E binding protein, or proteinA, respectively, to the target recombinant protein.

Examples of suitable inducible non-fusion E. coli expression vectorsinclude pTrc (Amann et al. (1988) Gene 69:301-315) and pET 11d (Studieret al., Gene Expression Technology: Methods in Enzymology 185, AcademicPress, San Diego, Calif. (1990) 60-89). Target gene expression from thepTrc vector relies on host RNA polymerase transcription from a hybridtrp-lac fusion promoter. Target gene expression from the pET 11d vectorrelies on transcription from a T7 gn10-lac fusion promoter mediated by acoexpressed viral RNA polymerase (T7 gn1). This viral polymerase issupplied by host strains BL21(DE3) or HMS174(DE3) from a resident ëprophage harboring a T7 gn1 gene under the transcriptional control ofthe lacUV5 promoter.

One strategy to maximize recombinant protein expression in E. coli is toexpress the protein in a bacterial having an impaired capacity toproteolytically cleave the recombinant protein (Gottesman, GeneExpression Technology: Methods in Enzymology 185, Academic Press, SanDiego, Calif. (1990) 119-128). Another strategy is to alter the nucleicacid sequence of the nucleic acid to be inserted into an expressionvector so that the individual codons for each amino acid are thosepreferentially utilized in E. coli (Wada et al. (1992) Nucleic AcidsRes. 20:2111-2118). Such alteration of nucleic acid sequences of theinvention can be carried out by standard DNA synthesis techniques.

In another embodiment, the CARD-9, CARD-10, or CARD-11 expression vectoris a yeast expression vector. Examples of vectors for expression inyeast S. cerivisae include pYepSec1 (Baldari et al. (1987) EMBO J.6:229-234), pMFa (Kurjan and Herskowitz, (1982) Cell 30:933-943), pJRY88(Schultz et al. (1987) Gene 54:113-123), pYES2 (Invitrogen Corporation,San Diego, Calif.), pGBT9 (Clontech, Palo Alto, Calif.), pGAD10(Clontech, Palo Alto, Calif.), pYADE4 and pYGAE2 and pYPGE2 (Brunelliand Pall (1993) Yeast 9:1299-1308), pYPGE15 (Brunelli and Pall (1993)Yeast 9:1309-1318), pACT11 (Dr. S. E. Elledge, Baylor College ofMedicine), and picZ (InVitrogen Corp, San Diego, Calif.).

Alternatively, CARD-9, CARD-10, or CARD-11 can be expressed in insectcells using baculovirus expression vectors. Baculovirus vectorsavailable for expression of proteins in cultured insect cells (e.g., Sf9cells) include the pAc series (Smith et al. (1983) Mol. Cell Biol.3:2156-2165) and the pVL series (Lucklow and Summers (1989) Virology170:31-39).

In yet another embodiment, a nucleic acid of the invention is expressedin mammalian cells using a mammalian expression vector. Examples ofmammalian expression vectors include pCDM8 (Seed (1987) Nature 329:840),pCI (Promega), and pMT2PC (Kaufman et al. (1987) EMBO J. 6:187-195).When used in mammalian cells, the expression vector's control functionsare often provided by viral regulatory elements. For example, commonlyused promoters are derived from polyoma, Adenovirus 2, cytomegalovirusand Simian Virus 40. For other suitable expression systems for bothprokaryotic and eukaryotic cells see chapters 16 and 17 of Sambrook etal. (supra).

In another embodiment, the recombinant mammalian expression vector iscapable of directing expression of the nucleic acid preferentially in aparticular cell type (e.g., tissue-specific regulatory elements are usedto express the nucleic acid). Tissue-specific regulatory elements areknown in the art. Non-limiting examples of suitable tissue-specificpromoters include the albumin promoter (liver-specific; Pinkert et al.(1987) Genes Dev. 1:268-277), lymphoid-specific promoters (Calame andEaton (1988) Adv. Immunol. 43:235-275), in particular promoters of Tcell receptors (Winoto and Baltimore (1989) EMBO J. 8:729-733) andimmunoglobulins (Banerji et al. (1983) Cell 33:729-740; Queen andBaltimore (1983) Cell 33:741-748), neuron-specific promoters (e.g., theneurofilament promoter; Byrne and Ruddle (1989) Proc. Natl. Acad. Sci.USA 86:5473-5477), pancreas-specific promoters (Edlund et al. (1985)Science 230:912-916), and mammary gland-specific promoters (e.g., milkwhey promoter; U.S. Pat. No. 4,873,316 and European ApplicationPublication No. 264,166). Developmentally-regulated promoters are alsoencompassed, for example the murine hox promoters (Kessel and Gruss(1990) Science 249:374-379) and the a-fetoprotein promoter (Campes andTilghman (1989) Genes Dev. 3:537-546).

The invention further provides a recombinant expression vectorcomprising a DNA molecule of the invention cloned into the expressionvector in an antisense orientation. That is, the DNA molecule isoperatively linked to a regulatory sequence in a manner which allows forexpression (by transcription of the DNA molecule) of an RNA moleculewhich is antisense to CARD-9, CARD-10, or CARD-11 mRNA. Regulatorysequences operatively linked to a nucleic acid cloned in the antisenseorientation can be chosen which direct the continuous expression of theantisense RNA molecule in a variety of cell types, for instance viralpromoters and/or enhancers, or regulatory sequences can be chosen whichdirect constitutive, tissue specific or cell type specific expression ofantisense RNA. The antisense expression vector can be in the form of arecombinant plasmid, phagemid or attenuated virus in which antisensenucleic acids are produced under the control of a high efficiencyregulatory region, the activity of which can be determined by the celltype into which the vector is introduced. For a discussion of theregulation of gene expression using antisense genes see Weintraub et al.(Reviews—Trends in Genetics, Vol. 1(1) 1986).

Another aspect of the invention pertains to host cells into which arecombinant expression vector of the invention or isolated nucleic acidmolecule of the invention has been introduced. The terms “host cell” and“recombinant host cell” are used interchangeably herein. It isunderstood that such terms refer not only to the particular subject cellbut to the progeny or potential progeny of such a cell. Because certainmodifications may occur in succeeding generations due to either mutationor environmental influences, such progeny may not, in fact, be identicalto the parent cell, but are still included within the scope of the termas used herein.

A host cell can be any prokaryotic or eukaryotic cell. For example,CARD-9, CARD-10, or CARD-11 protein can be expressed in bacterial cellssuch as E. coli, insect cells, yeast or mammalian cells (such as Chinesehamster ovary cells (CHO) or COS cells). Other suitable host cells areknown to those skilled in the art.

Vector DNA or an isolated nucleic acid molecule of the invention can beintroduced into prokaryotic or eukaryotic cells via conventionaltransformation or transfection techniques. As used herein, the terms“transformation” and “transfection” are intended to refer to a varietyof art-recognized techniques for introducing foreign nucleic acid (e.g.,DNA) into a host cell, including calcium phosphate or calcium chlorideco-precipitation, DEAE-dextran-mediated transfection, lipofection, orelectroporation. Suitable methods for transforming or transfecting hostcells can be found in Sambrook, et al. (supra), and other laboratorymanuals.

For stable transfection of mammalian cells, it is known that, dependingupon the expression vector and transfection technique used, only a smallfraction of cells may integrate the foreign DNA into their genome. Insome cases vector DNA is retained by the host cell. In other cases thehost cell does not retain vector DNA and retains only an isolatednucleic acid molecule of the invention carried by the vector. In somecases, and isolated nucleic acid molecule of the invention is used totransform a cell without the use of a vector.

In order to identify and select these integrants, a gene that encodes aselectable marker (e.g., resistance to antibiotics) is generallyintroduced into the host cells along with the gene of interest.Preferred selectable markers include those which confer resistance todrugs, such as G418, hygromycin and methotrexate. Nucleic acid encodinga selectable marker can be introduced into a host cell on the samevector as that encoding CARD-9, CARD-10, or CARD-11 or can be introducedon a separate vector. Cells stably transfected with the introducednucleic acid can be identified by drug selection (e.g., cells that haveincorporated the selectable marker gene will survive, while the othercells die).

A host cell of the invention, such as a prokaryotic or eukaryotic hostcell in culture, can be used to produce (i.e., express) a CARD-9,CARD-10, or CARD-11 protein. Accordingly, the invention further providesmethods for producing CARD-9, CARD-10, or CARD-11 protein using the hostcells of the invention. In one embodiment, the method comprisesculturing the host cell of the invention (into which a recombinantexpression vector or isolated nucleic acid molecule encoding CARD-9,CARD-10, or CARD-11 has been introduced) in a suitable medium such thatCARD-9, CARD-10, or CARD-11 protein is produced. In another embodiment,the method further comprises isolating CARD-9, CARD-10, or CARD-11 fromthe medium or the host cell.

The host cells of the invention can also be used to produce nonhumantransgenic animals. For example, in one embodiment, a host cell of theinvention is a fertilized oocyte or an embryonic stem cell into whichCARD-9, CARD-10, or CARD-11-coding sequences have been introduced. Suchhost cells can then be used to create non-human transgenic animals inwhich exogenous CARD-9, CARD-10, or CARD-11 sequences have beenintroduced into their genome or homologous recombinant animals in whichendogenous CARD-9, CARD-10, or CARD-11 sequences have been altered. Suchanimals are useful for studying the function and/or activity of CARD-9,CARD-10, or CARD-11 and for identifying and/or evaluating modulators ofCARD-9, CARD-10, or CARD-11 activity. As used herein, a “transgenicanimal” is a non-human animal, preferably a mammal, more preferably arodent such as a rat or mouse, in which one or more of the cells of theanimal includes a transgene. Other examples of transgenic animalsinclude non-human primates, sheep, dogs, cows, goats, chickens,amphibians, etc. A transgene is exogenous DNA which is integrated intothe genome of a cell from which a transgenic animal develops and whichremains in the genome of the mature animal, thereby directing theexpression of an encoded gene product in one or more cell types ortissues of the transgenic animal. As used herein, an “homologousrecombinant animal” is a non-human animal, preferably a mammal, morepreferably a mouse, in which an endogenous CARD-9, CARD-10, or CARD-11gene has been altered by homologous recombination between the endogenousgene and an exogenous DNA molecule introduced into a cell of the animal,e.g., an embryonic cell of the animal, prior to development of theanimal.

A transgenic animal of the invention can be created by introducingCARD-9, CARD-10, or CARD-11-encoding nucleic acid into the malepronuclei of a fertilized oocyte, e.g., by microinjection, retroviralinfection, and allowing the oocyte to develop in a pseudopregnant femalefoster animal. The CARD-9, CARD-10, or CARD-11 cDNA sequence, e.g., thatof SEQ ID NO: 1, SEQ ID NO: 3, SEQ ID NO: 4, SEQ ID NO: 6, SEQ ID NO: 7,SEQ ID NO: 9, SEQ ID NO: 10, or SEQ ID NO: 12 can be introduced as atransgene into the genome of a non-human animal. Alternatively, anonhuman homolog or ortholog of the human CARD-9, CARD-10, or CARD-11gene, such as a mouse CARD-9, CARD-10, or CARD-11 gene, can be isolatedbased on hybridization to the human CARD-9, CARD-10, or CARD-11 cDNA andused as a transgene. Intronic sequences and polyadenylation signals canalso be included in the transgene to increase the efficiency ofexpression of the transgene. A tissue-specific regulatory sequence(s)can be operably linked to the CARD-9, CARD-10, or CARD-11 transgene todirect expression of CARD-9, CARD-10, or CARD-11 protein to particularcells. Methods for generating transgenic animals via embryo manipulationand microinjection, particularly animals such as mice, have becomeconventional in the art and are described, for example, in U.S. Pat.Nos. 4,736,866 and 4,870,009, U.S. Pat. No. 4,873,191 and in Hogan,Manipulating the Mouse Embryo, (Cold Spring Harbor Laboratory Press,Cold Spring Harbor, N.Y., 1986). Similar methods are used for productionof other transgenic animals. A transgenic founder animal can beidentified based upon the presence of the CARD-9, CARD-10, or CARD-11transgene in its genome and/or expression of CARD-9, CARD-10, or CARD-11mRNA in tissues or cells of the animals. A transgenic founder animal canthen be used to breed additional animals carrying the transgene.Moreover, transgenic animals carrying a transgene encoding CARD-9,CARD-10, or CARD-11 can further be bred to other transgenic animalscarrying other transgenes.

To create an homologous recombinant animal, a vector is prepared whichcontains at least a portion of a CARD-9, CARD-10, or CARD-11 gene (e.g.,a human or a non-human homolog of the CARD-9, CARD-10, or CARD-11 gene,e.g., a murine CARD-9, CARD-10, or CARD-11 gene) into which a deletion,addition or substitution has been introduced to thereby alter, e.g.,functionally disrupt, the CARD-9, CARD-10, or CARD-11 gene. In anembodiment, the vector is designed such that, upon homologousrecombination, the endogenous CARD-9, CARD-10, or CARD-11 gene isfunctionally disrupted (i.e., no longer encodes a functional protein;also referred to as a “knock out” vector). Alternatively, the vector canbe designed such that, upon homologous recombination, the endogenousCARD-9, CARD-10, or CARD-11 gene is mutated or otherwise altered butstill encodes functional protein (e.g., the upstream regulatory regioncan be altered to thereby alter the expression of the endogenous CARD-9,CARD-10, or CARD-11 protein). In the homologous recombination vector,the altered portion of the CARD-9, CARD-10, or CARD-11 gene is flankedat its 5′ and 3′ ends by additional nucleic acid of the CARD-9, CARD-10,or CARD-11 gene to allow for homologous recombination to occur betweenthe exogenous CARD-9, CARD-10, or CARD-11 gene carried by the vector andan endogenous CARD-9, CARD-10, or CARD-11 gene in an embryonic stemcell. The additional flanking CARD-9, CARD-10, or CARD-11 nucleic acidis of sufficient length for successful homologous recombination with theendogenous gene. Typically, several kilobases of flanking DNA (both atthe 5′ and 3′ ends) are included in the vector (see, e.g., Thomas andCapecchi (1987) Cell 51:503 for a description of homologousrecombination vectors). The vector is introduced into an embryonic stemcell line (e.g., by electroporation) and cells in which the introducedCARD-9, CARD-10, or CARD-11 gene has homologously recombined with theendogenous CARD-9, CARD-10, or CARD-11 gene are selected (see, e.g., Liet al. (1992) Cell 69:915). The selected cells are then injected into ablastocyst of an animal (e.g., a mouse) to form aggregation chimeras(see, e.g., Bradley in Teratocarcinomas and Embryonic Stem Cells: APractical Approach, Robertson, ed. (IRL, Oxford, 1987) pp. 113-152). Achimeric embryo can then be implanted into a suitable pseudopregnantfemale foster animal and the embryo brought to term. Progeny harboringthe homologously recombined DNA in their germ cells can be used to breedanimals in which all cells of the animal contain the homologouslyrecombined DNA by germline transmission of the transgene. Methods forconstructing homologous recombination vectors and homologous recombinantanimals are described further in Bradley (1991) Current Opinion inBio/Technology 2:823-829 and in PCT Publication Nos. WO 90/11354, WO91/01140, WO 92/0968, and WO 93/04169.

In another embodiment, transgenic non-humans animals can be producedwhich contain selected systems which allow for regulated expression ofthe transgene. One example of such a system is the cre/loxP recombinasesystem of bacteriophage P1. For a description of the cre/loxPrecombinase system, see, e.g., Lakso et al. (1992) Proc. Natl. Acad.Sci. USA 89:6232-6236. Another example of a recombinase system is theFLP recombinase system of Saccharomyces cerevisiae (O'Gorman et al.(1991) Science 251:1351-1355. If a cre/loxP recombinase system is usedto regulate expression of the transgene, animals containing transgenesencoding both the Cre recombinase and a selected protein are required.Such animals can be provided through the construction of “double”transgenic animals, e.g., by mating two transgenic animals, onecontaining a transgene encoding a selected protein and the othercontaining a transgene encoding a recombinase.

Clones of the non-human transgenic animals described herein can also beproduced according to the methods described in Wilmut et al. (1997)Nature 385:810-813 and PCT Publication Nos. WO 97/07668 and WO 97/07669.In brief, a cell, e.g., a somatic cell, from the transgenic animal canbe isolated and induced to exit the growth cycle and enter Go phase. Thequiescent cell can then be fused, e.g., through the use of electricalpulses, to an enucleated oocyte from an animal of the same species fromwhich the quiescent cell is isolated. The reconstructed oocyte is thencultured such that it develops to morula or blastocyte and thentransferred to pseudopregnant female foster animal. The offspring borneof this female foster animal will be a clone of the animal from whichthe cell, e.g., the somatic cell, is isolated.

In another embodiment, the expression characteristics of an endogenousCARD-9, CARD-10, or CARD-11 gene within a cell line or microorganism maybe modified by inserting a heterologous DNA regulatory element into thegenome of a stable cell line or cloned microorganism such that theinserted regulatory element is operatively linked with the endogenousCARD-9, CARD-10, or CARD-11 gene. For example, an endogenous CARD-9,CARD-10, or CARD-11 which is normally “transcriptionally silent,” i.e. aCARD-9, CARD-10, or CARD-11 gene which is normally not expressed, or isexpressed only at very low levels in a cell line or microorganism, maybe activated by inserting a regulatory element which is capable ofpromoting the expression of a normally expressed gene product in thatcell line or microorganism. Alternatively, a transcriptionally silent,endogenous CARD-9, CARD-10, or CARD-11 gene may be activated byinsertion of a promiscuous regulatory element that works across celltypes.

A heterologous regulatory element may be inserted into a stable cellline or cloned microorganism, such that it is operatively linked with anendogenous CARD-9, CARD-10, or CARD-11 gene, using techniques, such astargeted homologous recombination, which are well known to those ofskill in the art, and described e.g., in Chappel, U.S. Pat. No.5,272,071; PCT publication No. WO 91/06667, published May 16, 1991.

V. Pharmaceutical Compositions

The CARD-9, CARD-10, or CARD-11 nucleic acid molecules, CARD-9, CARD-10,or CARD-11 proteins, and anti-CARD-9, CARD-10, or CARD11 antibodies(also referred to herein as “active compounds”) of the invention can beincorporated into pharmaceutical compositions suitable foradministration. Such compositions typically comprise the nucleic acidmolecule, protein, or antibody and a pharmaceutically acceptablecarrier. As used herein the language “pharmaceutically acceptablecarrier” is intended to include any and all solvents, dispersion media,coatings, antibacterial and antifungal agents, isotonic and absorptiondelaying agents, and the like, compatible with pharmaceuticaladministration. The use of such media and agents for pharmaceuticallyactive substances is well known in the art. Except insofar as anyconventional media or agent is incompatible with the active compound,use thereof in the compositions is contemplated. Supplementary activecompounds can also be incorporated into the compositions.

The invention includes methods for preparing pharmaceutical compositionsfor modulating the expression or activity of a polypeptide or nucleicacid of the invention. Such methods comprise formulating apharmaceutically acceptable carrier with an agent which modulatesexpression or activity of a polypeptide or nucleic acid of theinvention. Such compositions can further include additional activeagents. Thus, the invention further includes methods for preparing apharmaceutical composition by formulating a pharmaceutically acceptablecarrier with an agent which modulates expression or activity of apolypeptide or nucleic acid of the invention and one or more additionalactive compounds.

The agent which modulates expression or activity may, for example, be asmall molecule. For example, such small molecules include peptides,peptidomimetics, amino acids, amino acid analogs, polynucleotides,polynucleotide analogs, nucleotides, nucleotide analogs, organic orinorganic compounds (i.e., including heteroorganic and organometalliccompounds) having a molecular weight less than about 10,000 grams permole, organic or inorganic compounds having a molecular weight les thanabout 5,000 grams per mole, organic or inorganic compounds having amolecular weight less than about 1,000 grams per mole, organic orinorganic compounds having a molecular weight less than about 500 gramsper mole, and salts, esters, and other pharmaceutically acceptable formsof such compounds. It is understood that appropriate doses of smallmolecule agents depends upon a number of factors within the ken of theordinarily skilled physician, veterinarian, or researcher. The dose(s)of the small molecule will vary, for example, depending upon theidentity, size, and condition of the subject or sample being treated,further depending upon the route by which the composition is to beadministered, if applicable, and the effect which the practitionerdesires the small molecule to have upon the nucleic acid or polypeptideof the invention. Exemplary doses include milligram or microgram amountsof the small molecule per kilogram of subject or sample weight (e.g.,about 1 microgram per kilogram to about 500 milligrams per kilogram,about 100 micrograms per kilogram to about 5 milligrams per kilogram, orabout 1 microgram per kilogram to about 50 micrograms per kilogram. Itis furthermore understood that appropriate doses of a small moleculedepend upon the potency of the small molecule with respect to theexpression or activity to be modulated. Such appropriate doses may bedetermined using the assays described herein. When one or more of thesesmall molecules is to be administered to an animal (e.g., a human) inorder to modulate expression or activity of a polypeptide or nucleicacid of the invention, a physician, veterinarian, or researcher may, forexample, prescribe a relatively low dose at first, subsequentlyincreasing the dose until an appropriate response is obtained. Inaddition, it is understood that the specific dose level for anyparticular subject will depend upon a variety of factors including theactivity of the specific compound employed, the age, body weight,general health, gender, and diet of the subject, the time ofadministration, the route of administration, the rate of excretion, anydrug combination, and the degree of expression or activity to bemodulated.

A pharmaceutical composition of the invention is formulated to becompatible with its intended route of administration. Examples of routesof administration include parenteral, e.g., intravenous, intradermal,subcutaneous, oral (e.g., inhalation), transdermal (topical),transmucosal, and rectal administration. Solutions or suspensions usedfor parenteral, intradermal, or subcutaneous application can include thefollowing components: a sterile diluent such as water for injection,saline solution, fixed oils, polyethylene glycols, glycerine, propyleneglycol or other synthetic solvents; antibacterial agents such as benzylalcohol or methyl parabens; antioxidants such as ascorbic acid or sodiumbisulfite; chelating agents such as ethylenediaminetetraacetic acid;buffers such as acetates, citrates or phosphates and agents for theadjustment of tonicity such as sodium chloride or dextrose. The pH canbe adjusted with acids or bases, such as hydrochloric acid or sodiumhydroxide. The parenteral preparation can be enclosed in ampoules,disposable syringes or multiple dose vials made of glass or plastic.

Pharmaceutical compositions suitable for injectable use include sterileaqueous solutions (where water soluble) or dispersions and sterilepowders for the extemporaneous preparation of sterile injectablesolutions or dispersion. For intravenous administration, suitablecarriers include physiological saline, bacteriostatic water, CremophorEL (BASF; Parsippany, N.J.) or phosphate buffered saline (PBS). In allcases, the composition must be sterile and should be fluid to the extentthat easy syringability exists. It must be stable under the conditionsof manufacture and storage and must be preserved against thecontaminating action of microorganisms such as bacteria and fungi. Thecarrier can be a solvent or dispersion medium containing, for example,water, ethanol, polyol (for example, glycerol, propylene glycol, andliquid polyetheylene glycol, and the like), and suitable mixturesthereof. The proper fluidity can be maintained, for example, by the useof a coating such as lecithin, by the maintenance of the requiredparticle size in the case of dispersion and by the use of surfactants.Prevention of the action of microorganisms can be achieved by variousantibacterial and antifungal agents, for example, parabens,chlorobutanol, phenol, ascorbic acid, thimerosal, and the like. In manycases, it will be preferable to include isotonic agents, for example,sugars, polyalcohols such as mannitol, sorbitol, sodium chloride in thecomposition. Prolonged absorption of the injectable compositions can bebrought about by including in the composition an agent which delaysabsorption, for example, aluminum monostearate and gelatin.

Sterile injectable solutions can be prepared by incorporating the activecompound (e.g., a CARD-9, CARD-10, or CARD-11 protein or anti-CARD-9,CARD-10, or CARD-11 antibody) in the required amount in an appropriatesolvent with one or a combination of ingredients enumerated above, asrequired, followed by filtered sterilization. Generally, dispersions areprepared by incorporating the active compound into a sterile vehiclewhich contains a basic dispersion medium and the required otheringredients from those enumerated above. In the case of sterile powdersfor the preparation of sterile injectable solutions, the preferredmethods of preparation are vacuum drying and freeze-drying which yieldsa powder of the active ingredient plus any additional desired ingredientfrom a previously sterile-filtered solution thereof.

Oral compositions generally include an inert diluent or an ediblecarrier. They can be enclosed in gelatin capsules or compressed intotablets. For the purpose of oral therapeutic administration, the activecompound can be incorporated with excipients and used in the form oftablets, troches, or capsules. Oral compositions can also be preparedusing a fluid carrier for use as a mouthwash, wherein the compound inthe fluid carrier is applied orally and swished and expectorated orswallowed. Pharmaceutically compatible binding agents, and/or adjuvantmaterials can be included as part of the composition. The tablets,pills, capsules, troches and the like can contain any of the followingingredients, or compounds of a similar nature: a binder such asmicrocrystalline cellulose, gum tragacanth or gelatin; an excipient suchas starch or lactose, a disintegrating agent such as alginic acid,Primogel, or corn starch; a lubricant such as magnesium stearate orSterotes; a glidant such as colloidal silicon dioxide; a sweeteningagent such as sucrose or saccharin; or a flavoring agent such aspeppermint, methyl salicylate, or orange flavoring. For administrationby inhalation, the compounds are delivered in the form of an aerosolspray from pressured container or dispenser which contains a suitablepropellant, e.g., a gas such as carbon dioxide, or a nebulizer.

Systemic administration can also be by transmucosal or transdermalmeans. For transmucosal or transdermal administration, penetrantsappropriate to the barrier to be permeated are used in the formulation.Such penetrants are generally known in the art, and include, forexample, for transmucosal administration, detergents, bile salts, andfusidic acid derivatives. Transmucosal administration can beaccomplished through the use of nasal sprays or suppositories. Fortransdermal administration, the active compounds are formulated intoointments, salves, gels, or creams as generally known in the art.

The compounds can also be prepared in the form of suppositories (e.g.,with conventional suppository bases such as cocoa butter and otherglycerides) or retention enemas for rectal delivery.

In one embodiment, the active compounds are prepared with carriers thatwill protect the compound against rapid elimination from the body, suchas a controlled release formulation, including implants andmicroencapsulated delivery systems. Biodegradable, biocompatiblepolymers can be used, such as ethylene vinyl acetate, polyanhydrides,polyglycolic acid, collagen, polyorthoesters, and polylactic acid.Methods for preparation of such formulations will be apparent to thoseskilled in the art. The materials can also be obtained commercially fromAlza Corporation and Nova Pharmaceuticals, Inc. Liposomal suspensions(including liposomes targeted to infected cells with monoclonalantibodies to viral antigens) can also be used as pharmaceuticallyacceptable carriers. These can be prepared according to methods known tothose skilled in the art, for example, as described in U.S. Pat. No.4,522,811.

It is especially advantageous to formulate oral or parenteralcompositions in dosage unit form for ease of administration anduniformity of dosage. Dosage unit form as used herein refers tophysically discrete units suited as unitary dosages for the subject tobe treated; each unit containing a predetermined quantity of activecompound calculated to produce the desired therapeutic effect inassociation with the required pharmaceutical carrier. The specificationfor the dosage unit forms of the invention are dictated by and directlydependent on the unique characteristics of the active compound and theparticular therapeutic effect to be achieved, and the limitationsinherent in the art of compounding such an active compound for thetreatment of individuals.

For antibodies, the preferred dosage is 0.1 mg/kg to 100 mg/kg of bodyweight (generally 10 mg/kg to 20 mg/kg). If the antibody is to act inthe brain, a dosage of 50 mg/kg to 100 mg/kg is usually appropriate.Generally, partially human antibodies and fully human antibodies have alonger half-life within the human body than other antibodies.Accordingly, lower dosages and less frequent administration is oftenpossible. Modifications such as lipidation can be used to stabilizeantibodies and to enhance uptake and tissue penetration (e.g., into thebrain). A method for lipidation of antibodies is described by Cruikshanket al. ((1997) J. Acquired Immune Deficiency Syndromes and HumanRetrovirology 14:193).

The nucleic acid molecules of the invention can be inserted into vectorsand used as gene therapy vectors. Gene therapy vectors can be deliveredto a subject by, for example, intravenous injection, localadministration (U.S. Pat. No. 5,328,470) or by stereotactic injection(see, e.g., Chen et al. (1994) Proc. Natl. Acad. Sci. USA 91:3054-3057).The pharmaceutical preparation of the gene therapy vector can includethe gene therapy vector in an acceptable diluent, or can comprise a slowrelease matrix in which the gene delivery vehicle is imbedded.Alternatively, where the complete gene delivery vector can be producedintact from recombinant cells, e.g. retroviral vectors, thepharmaceutical preparation can include one or more cells which producethe gene delivery system.

The pharmaceutical compositions can be included in a container, pack, ordispenser together with instructions for administration.

VI. Uses and Methods of the Invention

The nucleic acid molecules, proteins, protein homologues, and antibodiesdescribed herein can be used in one or more of the following methods: a)screening assays; b) detection assays (e.g., chromosomal mapping, tissuetyping, forensic biology), c) predictive medicine (e.g., diagnosticassays, prognostic assays, monitoring clinical trials, andpharmacogenomics); and d) methods of treatment (e.g., therapeutic andprophylactic). A CARD-9, CARD-10, or CARD-11 protein interacts withother cellular proteins and can thus be used for (i) regulation ofcellular proliferation; (ii) regulation of cellular differentiation; and(iii) regulation of cell survival. The isolated nucleic acid moleculesof the invention can be used to express CARD-9, CARD-10, or CARD-11protein (e.g., via a recombinant expression vector in a host cell ingene therapy applications), to detect CARD-9, CARD-10, or CARD-11 mRNA(e.g., in a biological sample) or a genetic lesion in a CARD-9, CARD-10,or CARD-11 gene, and to modulate CARD-9, CARD-10, or CARD-11 activity.In addition, the CARD-9, CARD-10, or CARD-11 proteins can be used toscreen drugs or compounds which modulate the CARD-9, CARD-10, or CARD-11activity or expression as well as to treat disorders characterized byinsufficient or excessive production of CARD-9, CARD-10, or CARD-11protein or production of CARD-9, CARD-10, or CARD-11 protein forms whichhave decreased or aberrant activity compared to CARD-9, CARD-10, orCARD-11 wild type protein. In addition, the anti-CARD-9, CARD-10, orCARD-11 antibodies of the invention can be used to detect and isolateCARD-9, CARD-10, or CARD-11 proteins and modulate CARD-9, CARD-10, orCARD-11 activity.

This invention further pertains to novel agents identified by theabove-described screening assays and uses thereof for treatments asdescribed herein.

A. Screening Assays

The invention provides a method (also referred to herein as a “screeningassay”) for identifying modulators, i.e., candidate or test compounds oragents (e.g., peptides, peptidomimetics, small molecules or other drugs)which bind to CARD-9, CARD-10, or CARD-11 proteins or biologicallyactive portions thereof or have a stimulatory or inhibitory effect on,for example, CARD-9, CARD-10, or CARD-11 expression or CARD-9, CARD-10,or CARD-11 activity. Examples of biologically active portions of CARD-9include: amino acids 7-98 encoding a CARD domain; amino acids 140-416encoding a coiled-coil domain; amino acids 197-213 encoding anindole-3-glycerol phosphate synthase homology region; and amino acids285-338 encoding a cysteine rich repeat homology region. Examples ofbiologically active portions of human CARD-10 include: amino acids23-123 encoding a CARD domain; amino acids 147-457 encoding acoiled-coil domain; amino acids 704-772 encoding an SH3 domain; aminoacids 830-1032 encoding a GUK domain; and amino acids 366-398 encoding atropomyosin domain. Examples of biologically active portions of humanCARD-11 include: amino acids 6-112 encoding a CARD domain; amino acids130-431 encoding a coiled-coil domain; amino acids 635-748 encoding aPDZ domain; amino acids 766-834 encoding an SH3 domain; and amino acids882-1147 encoding a GUK domain.

Among the screening assays provided by the invention are screening toidentify molecules that prevent the dimerization of CARD-9, CARD-10, orCARD-11 and screening to identify molecules which block the binding of aCARD containing polypeptide to CARD-9, CARD-10, or CARD-11. Screeningassays, e.g., dimerization assays, can employ full-length CARD-9,CARD-10, or CARD-11 or a portion of CARD-9, CARD-10, or CARD-11, e.g.,the CARD domain, the coiled-coil domain, the PDZ domain, the SH3 domain,or the GUK domain.

Screening assays can be used to identify molecules which modulate aCARD-9, CARD-10, or CARD-11 mediated increase in transcription of geneshaving an AP-1 or NF-κB binding site. For example, expression of areporter gene under the control of NF-κB (or AP-1) is measured in thepresence and absence of a candidate molecule and in the presence andabsence of CARD-9, CARD-10, or CARD-11 to identify those molecules whichalter expression of the reporter in a CARD-9, CARD-10, or CARD-11dependent manner. In addition, screening assays can be used to identifymolecules that modulate a CARD-9, CARD-10, or CARD-11 mediated increasein CHOP phosphorylation. For example, the expression of a reporter geneunder the control of CHOP is measured in the presence and absence of acandidate small molecule and in the presence and absence of CARD-9,CARD-10, or CARD-11 to identify those molecules that alter expression ofthe reporter in a CARD-9, CARD-10, or CARD-11 dependent manner. Ascreening assay can be carried out to identify molecules which modulatethe CARD-9, CARD-10, or CARD-11 mediated increase in CHOPphosphorylation. For example, CHOP phosphorylation is measured in thepresence and absence of a candidate molecule and in the presence andabsence of CARD-9, CARD-10, or CARD-11. Phosphorylation of CHOP can bemeasured using an antibody which binds to phosphorylated CHOP, but notto non-phosphorylated CHOP.

Screening assays can also be used to identify molecules that modulateactivity mediated by a domain of CARD-9, CARD-10, or CARD-11. Forexample, enzymatic activity mediated by the GUK of CARD-10 or CARD-11may be measured by a GTP binding assay. Test compounds or agents may beevaluated for their ability to either increase or decrease theGTP-binding ability of the GUK domain of CARD-10 or CARD-11.

In one embodiment, the invention provides assays for screening candidateor test compounds which bind to or modulate the activity of a CARD-9,CARD-10, or CARD-11 proteins or polypeptides or biologically activeportions thereof. The test compounds of the present invention can beobtained using any of the numerous approaches in combinatorial librarymethods known in the art, including: biological libraries; spatiallyaddressable parallel solid phase or solution phase libraries; syntheticlibrary methods requiring deconvolution; the “one-bead one-compound”library method; and synthetic library methods using affinitychromatography selection. The biological library approach is limited topeptide libraries, while the other four approaches are applicable topeptide, non-peptide oligomer or small molecule libraries of compounds(Lam (1997) Anticancer Drug Des. 12:145). Examples of methods for thesynthesis of molecular libraries can be found in the art, for examplein: DeWitt et al. (1993) Proc. Natl. Acad. Sci. U.S.A. 90:6909; Erb etal. (1994) Proc. Natl. Acad. Sci. USA 91:11422; Zuckermann et al.(1994). J. Med. Chem. 37:2678; Cho et al. (1993) Science 261:1303;Carrell et al. (1994) Angew. Chem. Int. Ed. Engl. 33:2059; Carell et al.(1994) Angew. Chem. Int. Ed. Engl. 33:2061; and Gallop et al. (1994) J.Med. Chem. 37:1233.

Libraries of compounds may be presented in solution (e.g., Houghten(1992) Bio/Techniques 13:412-421), or on beads (Lam (1991) Nature354:82-84), chips (Fodor (1993) Nature 364:555-556), bacteria (U.S. Pat.No. 5,223,409), spores (U.S. Pat. Nos. 5,571,698; 5,403,484; and5,223,409), plasmids (Cull et al. (1992) Proc. Natl. Acad. Sci. USA89:1865-1869) or on phage (Scott and Smith (1990) Science 249:386-390;Devlin (1990) Science 249:404-406; Cwirla et al. (1990) Proc. Natl.Acad. Sci. 87:6378-6382; and Felici (1991) J. Mol. Biol. 222:301-310).

In one embodiment, an assay is one in which a polypeptide of theinvention, or a biologically active portion thereof, is contacted with atest compound and the ability of the test compound to bind to thepolypeptide determined. Determining the ability of the test compound tobind to the polypeptide can be accomplished, for example, by couplingthe test compound with a radioisotope or enzymatic label such thatbinding of the test compound to the polypeptide or biologically activeportion thereof can be determined by detecting the labeled compound in acomplex. For example, test compounds can be labeled with ¹²⁵I, ³⁵S, ¹⁴C,or ³H, either directly or indirectly, and the radioisotope detected bydirect counting of radioemmission or by scintillation counting.Alternatively, test compounds can be enzymatically labeled with, forexample, horseradish peroxidase, alkaline phosphatase, or luciferase,and the enzymatic label detected by determination of conversion of anappropriate substrate to product.

Determining the ability of the test compound to modulate the activity ofCARD-9, CARD-10, or CARD-11 or a biologically active portion thereof canbe accomplished, for example, by determining the ability of the CARD-9,CARD-10, or CARD-11 protein to bind to or interact with a CARD-9,CARD-10, or CARD-11 target molecule. As used herein, a “target molecule”is a molecule with which a CARD-9, CARD-10, or CARD-11 protein binds orinteracts in nature, for example, a molecule associated with theinternal surface of a cell membrane or a cytoplasmic molecule. A CARD-9,CARD-10, or CARD-11 target molecule can be a non-CARD-9, CARD-10, orCARD-11 molecule or a CARD-9, CARD-10, or CARD-11 protein or polypeptideof the present invention. In one embodiment, a CARD-9, CARD-10, orCARD-11 target molecule is a component of an apoptotic signaltransduction pathway. The target, for example, can be a secondintracellular protein which has catalytic activity or a protein whichfacilitates the association of downstream signaling molecules withCARD-9, CARD-10, or CARD-11.

Determining the ability of the test compound to modulate the activity ofCARD-9, CARD-10, or CARD-11 or a biologically active portion thereof canbe accomplished, for example, by determining the ability of the CARD-9,CARD-10, or CARD-11 protein to bind to or interact with any of thespecific proteins listed in the previous paragraph as CARD-9, CARD-10,or CARD-11 target molecules. In another embodiment, CARD-9, CARD-10, orCARD-11 target molecules include all proteins that bind to a CARD-9,CARD-10, or CARD-11 protein or a fragment thereof in a two-hybrid systembinding assay which can be used without undue experimentation to isolatesuch proteins from cDNA or genomic two-hybrid system libraries. Thebinding assays described in this section can be cell-based or cell free(described subsequently).

Determining the ability of the CARD-9, CARD-10, or CARD-11 protein tobind to or interact with a CARD-9, CARD-10, or CARD-11 target moleculecan be accomplished by one of the methods described above fordetermining direct binding. In an embodiment, determining the ability ofthe CARD-9, CARD-10, or CARD-11 protein to bind to or interact with aCARD-9, CARD-10, or CARD-11 target molecule can be accomplished bydetermining the activity of the target molecule. For example, theactivity of the target molecule can be determined by detecting inductionof a cellular second messenger of the target (e.g., intracellular Ca²⁺,diacylglycerol, IP3, etc.), detecting catalytic/enzymatic activity ofthe target on an appropriate substrate, detecting the induction of areporter gene (e.g., a CARD-9, CARD-10, or CARD-11-responsive regulatoryelement operatively linked to a nucleic acid encoding a detectablemarker, e.g. luciferase), or detecting a cellular response, for example,cell survival, cellular differentiation, or cell proliferation. Theactivity of a target molecule can be monitored by assaying the caspase9-mediated apoptosis cellular response or caspase 9 enzymatic activity.In addition, and in another embodiment, genes induced by CARD-9,CARD-10, or CARD-11 expression can be identified by expressing CARD-9,CARD-10, or CARD-11 in a cell line and conducting a transcriptionalprofiling experiment wherein the mRNA expression patterns of the cellline transformed with an empty expression vector and the cell linetransformed with a CARD-9, CARD-10, or CARD-11 expression vector arecompared. The promoters of genes induced by CARD-9, CARD-10, or CARD-11expression can be operatively linked to reporter genes suitable forscreening such as luciferase, secreted alkaline phosphatase, orbeta-galactosidase and the resulting constructs could be introduced intoappropriate expression vectors. A recombinant cell line containingCARD-9, CARD-10, or CARD-11 and transfected with an expression vectorcontaining a CARD-9, CARD-10, or CARD-11 responsive promoter operativelylinked to a reporter gene can be used to identify test compounds thatmodulate CARD-9, CARD-10, or CARD-11 activity by assaying the expressionof the reporter gene in response to contacting the recombinant cell linewith test compounds. CARD-9, CARD-10, or CARD-11 agonists can beidentified as increasing the expression of the reporter gene and CARD-9,CARD-10, or CARD-11 antagonists can be identified as decreasing theexpression of the reporter gene.

In another embodiment of the invention, the ability of a test compoundto modulate the activity of CARD-9, CARD-10, or CARD-11, or biologicallyactive portions thereof can be determined by assaying the ability of thetest compound to modulate CARD-9, CARD-10, or CARD-11-dependent pathwaysor processes where the CARD-9, CARD-10, or CARD-11 target proteins thatmediate the CARD-9, CARD-10, or CARD-11 effect are known or unknown.Potential CARD-9, CARD-10, or CARD-11-dependent pathways or processesinclude, but are not limited to, the modulation of cellular signaltransduction pathways and their related second messenger molecules(e.g., intracellular Ca²+, diacylglycerol, IP3, cAMP etc.), cellularenzymatic activities, cellular responses (e.g., cell survival, cellulardifferentiation, or cell proliferation), or the induction or repressionof cellular or heterologous mRNAs or proteins. CARD-9, CARD-10, orCARD-11-dependent pathways or processes could be assayed by standardcell-based or cell free assays appropriate for the specific pathway orprocess under study. In another embodiment, cells cotransfected withCARD-9, CARD-10, or CARD-11 and a NF-kB luciferase reporter gene couldbe contacted with a test compound and test compounds that block CARD-9,CARD-10, or CARD-11 activity could be identified by their reduction ofCARD-9, CARD-10, or CARD-11-dependent NF-kB pathway luciferase reportergene expression. Test compounds that agonize CARD-9, CARD-10, or CARD-11would be expected to increase reporter gene expression. In anotherembodiment, CARD-9, CARD-10, or CARD-11 could be expressed in a cellline and the recombinant CARD-9, CARD-10, or CARD-11-expressing cellline could be contacted with a test compound. Test compounds thatinhibit CARD-9, CARD-10, or CARD-11 activity could be identified bytheir reduction of CARD-9, CARD-10, or CARD-11-depended NF-kB pathwaystimulation as measured by the assay of a NF-kB pathway reporter gene,NF-kB nuclear localization, I_(K)B phosphorylation or proteolysis, orother standard assays for NF-kB pathway activation known to thoseskilled in the art.

In yet another embodiment, an assay of the present invention is acell-free assay comprising contacting a CARD-9, CARD-10, or CARD-11protein or biologically active portion thereof with a test compound anddetermining the ability of the test compound to bind to theCARD-9,CARD-10, or CARD-11 protein or biologically active portionthereof. Binding of the test compound to the CARD-9, CARD-10, or CARD-11protein can be determined either directly or indirectly as describedabove. In one embodiment, a competitive binding assay includescontacting the CARD-9, CARD-10, or CARD-11 protein or biologicallyactive portion thereof with a compound known to bind CARD-9, CARD-10, orCARD-11 to form an assay mixture, contacting the assay mixture with atest compound, and determining the ability of the test compound tointeract with a CARD-9, CARD-10, or CARD-11 protein, wherein determiningthe ability of the test compound to interact with a CARD-9, CARD-10, orCARD-11 protein comprises determining the ability of the test compoundto preferentially bind to CARD-9, CARD-10, or CARD-11 or biologicallyactive portion thereof as compared to the known binding compound.

In another embodiment, an assay is a cell-free assay comprisingcontacting CARD-9, CARD-10, or CARD-11 protein or biologically activeportion thereof with a test compound and determining the ability of thetest compound to modulate (e.g., stimulate or inhibit) the activity ofthe CARD-9, CARD-10, or CARD-11 protein or biologically active portionthereof. Determining the ability of the test compound to modulate theactivity of CARD-9, CARD-10, or CARD-11 can be accomplished, forexample, by determining the ability of the CARD-9, CARD-10, or CARD-11protein to bind to or interact with a CARD-9, CARD-10, or CARD-11 targetmolecule, e.g., Bcl-10, by one of the methods described above fordetermining direct binding. In an alternative embodiment, determiningthe ability of the test compound to modulate the activity of CARD-9,CARD-10, or CARD-11 can be accomplished by determining the ability ofthe CARD-9, CARD-10, or CARD-11 protein to further modulate a CARD-9,CARD-10, or CARD-11 target molecule. For example, thecatalytic/enzymatic activity of the target molecule on an appropriatesubstrate can be determined as previously described.

In yet another embodiment, the cell-free assay comprises contacting theCARD-9, CARD-10, or CARD-11 protein or biologically active portionthereof with a known compound which binds CARD-9, CARD-10, or CARD-11 toform an assay mixture, contacting the assay mixture with a testcompound, and determining the ability of the test compound to interactwith a CARD-9, CARD-10, or CARD-11 protein, wherein determining theability of the test compound to interact with a CARD-9, CARD-10, orCARD-11 protein comprises determining the ability of the CARD-9,CARD-10, or CARD-11 protein to preferentially bind to or modulate theactivity of a CARD-9, CARD-10, or CARD-11 target molecule. The cell-freeassays of the present invention are amenable to use of either thesoluble form or a membrane-associated form of CARD-9, CARD-10, orCARD-11. A membrane-associated form of CARD-9, CARD-10, or CARD-11refers to CARD-9, CARD-10, or CARD-11 that interacts with amembrane-bound target molecule. In the case of cell-free assayscomprising the membrane-associated form of CARD-9, CARD-10, or CARD-11,it may be desirable to utilize a solubilizing agent such that themembrane-associated form of CARD-9, CARD-10, or CARD-11 is maintained insolution. Examples of such solubilizing agents include non-ionicdetergents such as n-octylglucoside, n-dodecylglucoside,n-dodecylmaltoside, octanoyl-N-methylglucamide,decanoyl-N-methylglucamide, Triton® X-100, Triton® X-114, Thesit®,Isotridecypoly(ethylene glycol ether)n,3-[(3-cholamidopropyl)dimethylamminio]-1-propane sulfonate (CHAPS),3-[(3-cholamidopropyl)dimethylamminio]-2-hydroxy-1-propane sulfonate(CHAPSO), or N-dodecyl=N,N-dimethyl-3-ammonio-1-propane sulfonate.

A variety of methods can be used to identify compounds which inhibit theinteraction between CARD-9, CARD-10, or CARD-11 and Bcl-10. Among thesemethods is the reverse two-hybrid screen (Huang and Schreiber (1997)Proc. Natl. Acad. Sci USA 94:13396-401; Vidal et al. and (1996) Proc.Natl. Acad. Sci. USA 93:10315-20; and Vidal et al. (1996) Proc. Natl.Acad. Sci. USA 93:10321-6). To create a high throughput assay, thereverse two hybrid screen can be combined with nanodroplet technology(Huang and Schreiber (1997) supra; and Borchardt et al. (1997) Chem. andBiol. 4:961-68). Nanodroplet technology employs 100-200 nl dropletswhich contain cells, defined media, and beads to which are attached testcompounds. The screening can take place in the nanodroplets, and thetest compounds can be attached to the beads so that their release can becontrolled photochemically.

A scintillation proximity assay can be used to identify molecules whichmodulate the interaction between Bcl-10 and CARD-9, CARD-10, or CARD-11.In a scintillation proximity assay designed to measure the interactionbetween two proteins, one of the two proteins is radioactively labeled,e.g., tritiated, and the other protein is not. The two interactingproteins are incubated in the presence and absence of a test compound.The unlabeled protein is captured by a solid support material, e.g., abead, impregnated with a fluorescer. The fraction of the radiolabeledprotein that binds to the captured unlabeled protein is in close enoughproximity to the solid support material to activate the fluorescer toproduce light energy. The vast majority of the radiolabeled protein thatdoes not bind to the unlabeled protein is too far from the solid supportmaterial to activate the fluorescer. Thus, the level of light energyproduced by the fluorescer is indicative of the amount of radiolabeledprotein bound to the unlabeled protein captured by the solid supportmaterial. Scintillation proximity assays are described in U.S. Pat. No.4,568,649.

In more than one embodiment of the above assay methods of the presentinvention, it may be desirable to immobilize either CARD-9, CARD-10, orCARD-11 or its target molecule to facilitate separation of complexedfrom uncomplexed forms of one or both of the proteins, as well as toaccommodate automation of the assay. Binding of a test compound toCARD-9, CARD-10, or CARD-11, or interaction of CARD-9, CARD-10, orCARD-11 with a target molecule in the presence and absence of acandidate compound, can be accomplished in any vessel suitable forcontaining the reactants. Examples of such vessels include microtitreplates, test tubes, and micro-centrifuge tubes. In one embodiment, afusion protein can be provided which adds a domain that allows one orboth of the proteins to be bound to a matrix. For example,glutathione-S-transferase/CARD-9, CARD-10, or CARD-11 fusion proteins orglutathione-S-transferase/target fusion proteins can be adsorbed ontoglutathione sepharose beads (Sigma Chemical; St. Louis, Mo.) orglutathione derivatized microtitre plates, which are then combined withthe test compound or the test compound and either the non-adsorbedtarget protein or CARD-9, CARD-10, or CARD-11 protein, and the mixtureincubated under conditions conducive to complex formation (e.g., atphysiological conditions for salt and pH). Following incubation, thebeads or microtitre plate wells are washed to remove any unboundcomponents, the matrix immobilized in the case of beads, complexdetermined either directly or indirectly, for example, as describedabove. Alternatively, the complexes can be dissociated from the matrix,and the level of CARD-9, CARD-10, or CARD-11 binding or activitydetermined using standard techniques. In an alternative embodiment, MYCor HA epitope tag CARD-9, CARD-10, or CARD-11 fusion proteins or MYC orHA epitope tag target fusion proteins can be adsorbed onto anti-MYC oranti-HA antibody coated microbeads or onto anti-MYC or anti-HA antibodycoated microtitre plates, which are then combined with the test compoundor the test compound and either the non-adsorbed target protein orCARD-9, CARD-10, or CARD-11 protein, and the mixture incubated underconditions conducive to complex formation (e.g., at physiologicalconditions for salt and pH). Following incubation, the beads ormicrotitre plate wells are washed to remove any unbound components, thematrix immobilized in the case of beads, complex determined eitherdirectly or indirectly, for example, as described above. Alternatively,the complexes can be dissociated from the matrix, and the level ofCARD-9, CARD-10, or CARD-11 binding or activity determined usingstandard techniques.

Other techniques for immobilizing proteins on matrices can also be usedin the screening assays of the invention. For example, CARD-9, CARD-10,or CARD-11 or its target molecule can be immobilized utilizingconjugation of biotin and streptavidin. Biotinylated CARD-9, CARD-10, orCARD-11 target molecules can be prepared from biotin-NHS(N-hydroxy-succinimide) using techniques well known in the art (e.g.,biotinylation kit, Pierce Chemicals; Rockford, Ill.), and immobilized inthe wells of streptavidin-coated 96 well plates (Pierce Chemical).Alternatively, antibodies reactive with CARD-9, CARD-10, or CARD-11 ortarget molecules but which do not interfere with binding of the proteinto its target molecule can be derivatized to the wells of the plate, andunbound target or protein trapped in the wells by antibody conjugation.Methods for detecting such complexes, in addition to those describedabove for the GST-immobilized complexes and epitope tag immobilizedcomplexes, include immunodetection of complexes using antibodiesreactive with the CARD-9, CARD-10, or CARD-11 or target molecule, aswell as enzyme-linked assays which rely on detecting an enzymaticactivity associated with the CARD-9, CARD-10, or CARD-11 or a targetmolecule.

In another embodiment, modulators of CARD-9, CARD-10, or CARD-11expression are identified in a method in which a cell is contacted witha candidate compound and the expression of the CARD-9, CARD-10, orCARD-11 promoter, mRNA or protein in the cell is determined. The levelof expression of CARD-9, CARD-10, or CARD-11 mRNA or protein in thepresence of the candidate compound is compared to the level ofexpression of CARD-9, CARD-10, or CARD-11 mRNA or protein in the absenceof the candidate compound. The candidate compound can then be identifiedas a modulator of CARD-9, CARD-10, or CARD-11 expression based on thiscomparison. For example, when expression of CARD-9, CARD-10, or CARD-11mRNA or protein is greater (statistically significantly greater) in thepresence of the candidate compound than in its absence, the candidatecompound is identified as a stimulator of CARD-9, CARD-10, or CARD-11mRNA or protein expression. Alternatively, when expression of CARD-9,CARD-10, or CARD-11 mRNA or protein is less (statistically significantlyless) in the presence of the candidate compound than in its absence, thecandidate compound is identified as an inhibitor of CARD-9, CARD-10, orCARD-11 mRNA or protein expression. The level of CARD-9, CARD-10, orCARD-11 mRNA or protein expression in the cells can be determined bymethods described herein for detecting CARD-9, CARD-10, or CARD-11 mRNAor protein. The activity of the CARD-9, CARD-10, or CARD-11 promoter canbe assayed by linking the CARD-9, CARD-10, or CARD-11 promoter to areporter gene such as luciferase, secreted alkaline phosphatase, orbeta-galactosidase and introducing the resulting construct into anappropriate vector, transfecting a host cell line, and measuring theactivity of the reporter gene in response to test compounds.

In yet another aspect of the invention, the CARD-9, CARD-10, or CARD-11proteins can be used as “bait proteins” in a two-hybrid assay (for adiscussion of a mammalian two-hybrid assay, see e.g., Hosfield and Chang(1999) Strategies Newsletter 2(2):62-65) or three hybrid assay (see,e.g., U.S. Pat. No. 5,283,317; Zervos et al. (1993) Cell 72:223-232;Madura et al. (1993) J. Biol. Chem. 268:12046-12054; Bartel et al.(1993) Bio/Techniques 14:920-924; Iwabuchi et al. (1993) Oncogene8:1693-1696; and PCT Publication No. WO 94/10300), to identify otherproteins, which bind to or interact with CARD-9, CARD-10, or CARD-11(“CARD-9, CARD-10, or CARD-11-binding proteins” or “CARD-9, CARD-10, orCARD-11-bp”) and modulate CARD-9, CARD-10, or CARD-11 activity. SuchCARD-9, CARD-10, or CARD-11-binding proteins are also likely to beinvolved in the propagation of signals by the CARD-9, CARD-10, orCARD-11 proteins as, for example, upstream or downstream elements of theCARD-9, CARD-10, or CARD-11 pathway.

The two-hybrid system is based on the modular nature of mosttranscription factors, which consist of separable DNA-binding andactivation domains. Briefly, the assay utilizes two different DNAconstructs. In one construct, the gene that codes for CARD-9, CARD-10,or CARD-11 is fused to a gene encoding the DNA binding domain of a knowntranscription factor (e.g., GAL-4). In the other construct, a DNAsequence, from a library of DNA sequences, that encodes an unidentifiedprotein (“prey” or “sample”) is fused to a gene that codes for theactivation domain of the known transcription factor. If the “bait” andthe “prey” proteins are able to interact, in vivo, forming a CARD-9,CARD-10, or CARD-11-dependent complex, the DNA-binding and activationdomains of the transcription factor are brought into close proximity.This proximity allows transcription of a reporter gene (e.g., LacZ)which is operably linked to a transcriptional regulatory site responsiveto the transcription factor. Expression of the reporter gene can bedetected and cell colonies containing the functional transcriptionfactor can be isolated and used to obtain the cloned gene which encodesthe protein which interacts with CARD-9, CARD-10, or CARD-11.

In an embodiment of the invention, the ability of a test compound tomodulate the activity of CARD-9, CARD-10, or CARD-11, or a biologicallyactive portion thereof can be determined by assaying the ability of thetest compound to block the binding of CARD-9, CARD-10, or CARD-11 to itstarget proteins in a yeast or mammalian two-hybrid system assay. Thisassay could be automated for high throughput drug screening purposes. Inanother embodiment of the invention, CARD-9, CARD-10, or CARD-11 and atarget protein could be configured in the reverse two-hybrid system(Vidal et al. (1996) Proc. Natl. Acad. Sci. USA 93:10321-6 and Vidal etal. (1996) Proc. Natl. Acad. Sci. USA 93:10315-20) designed specificallyfor efficient drug screening. In the reverse two-hybrid system,inhibition of a CARD-9, CARD-10, or CARD-11 physical interaction with atarget protein would result in induction of a reporter gene in contrastto the normal two-hybrid system where inhibition of CARD-9, CARD-10, orCARD-11 physical interaction with a target protein would lead toreporter gene repression. The reverse two-hybrid system is preferred fordrug screening because reporter gene induction is more easily assayedthan report gene repression.

Alternative embodiments of the invention are proteins found tophysically interact with proteins that bind to CARD-9, CARD-10, orCARD-11. CARD-9, CARD-10, or CARD-11 interactors could be configuredinto two-hybrid system baits and used in two-hybrid screens to identifyadditional members of the CARD-9, CARD-10, or CARD-11 pathway. Theinteractors of CARD-9, CARD-10, or CARD-11 interactors identified inthis way could be useful targets for therapeutic intervention in CARD-9,CARD-10, or CARD-11 related diseases and pathologies and an assay oftheir enzymatic or binding activity could be useful for theidentification of test compounds that modulate CARD-9, CARD-10, orCARD-11 activity.

This invention further pertains to novel agents identified by theabove-described screening assays and uses thereof for treatments asdescribed herein.

B. Detection Assays

Portions or fragments of the cDNA sequences identified herein (and thecorresponding complete gene sequences) can be used in numerous ways aspolynucleotide reagents. For example, these sequences can be used to:(i) map their respective genes on a chromosome; and, thus, locate generegions associated with genetic disease; (ii) identify an individualfrom a minute biological sample (tissue typing); and (iii) aid inforensic identification of a biological sample. These applications aredescribed in the subsections below.

1. Chromosome Mapping

Once the sequence (or a portion of the sequence) of a gene has beenisolated, this sequence can be used to map the location of the gene on achromosome. Accordingly, CARD-9, CARD-10, or CARD-11 nucleic acidmolecules described herein or fragments thereof, can be used to map thelocation of CARD-9, CARD-10, or CARD-11 genes on a chromosome. Themapping of the CARD-9, CARD-10, or CARD-11 sequences to chromosomes isan important first step in correlating these sequences with genesassociated with disease.

Briefly, CARD-9, CARD-10, or CARD-11 genes can be mapped to chromosomesby preparing PCR primers (preferably 15-25 bp in length) from theCARD-9, CARD-10, or CARD-11 sequences. Computer analysis of CARD-9,CARD-10, or CARD-11 sequences can be used to rapidly select primers thatdo not span more than one exon in the genomic DNA, thus complicating theamplification process. These primers can then be used for PCR screeningof somatic cell hybrids containing individual human chromosomes. Onlythose hybrids containing the human gene corresponding to the CARD-9,CARD-10, or CARD-11 sequences will yield an amplified fragment.

Somatic cell hybrids are prepared by fusing somatic cells from differentmammals (e.g., human and mouse cells). As hybrids of human and mousecells grow and divide, they gradually lose human chromosomes in randomorder, but retain the mouse chromosomes. By using media in which mousecells cannot grow, because they lack a particular enzyme, but humancells can, the one human chromosome that contains the gene encoding theneeded enzyme, will be retained. By using various media, panels ofhybrid cell lines can be established. Each cell line in a panel containseither a single human chromosome or a small number of human chromosomes,and a full set of mouse chromosomes, allowing easy mapping of individualgenes to specific human chromosomes. (D'Eustachio et al. (1983) Science220:919-924). Somatic cell hybrids containing only fragments of humanchromosomes can also be produced using human chromosomes withtranslocations and deletions.

PCR mapping of somatic cell hybrids is a rapid procedure for assigning aparticular sequence to a particular chromosome. Three or more sequencescan be assigned per day using a single thermal cycler. Using the CARD-9,CARD-10, or CARD-11 sequences to design oligonucleotide primers,sublocalization can be achieved with panels of fragments from specificchromosomes. Other mapping strategies which can similarly be used to mapa CARD-9, CARD-10, or CARD-11 sequence to its chromosome include in situhybridization (described in Fan et al. (1990) Proc. Natl. Acad. Sci. USA87:6223-27), pre-screening with labeled flow-sorted chromosomes, andpre-selection by hybridization to chromosome specific cDNA libraries.

Fluorescence in situ hybridization (FISH) of a DNA sequence to ametaphase chromosomal spread can further be used to provide a precisechromosomal location in one step. Chromosome spreads can be made usingcells whose division has been blocked in metaphase by a chemical likecolcemid that disrupts the mitotic spindle. The chromosomes can betreated briefly with trypsin, and then stained with Giemsa. A pattern oflight and dark bands develops on each chromosome, so that thechromosomes can be identified individually. The FISH technique can beused with a DNA sequence as short as 500 or 600 bases. However, cloneslarger than 1,000 bases have a higher likelihood of binding to a uniquechromosomal location with sufficient signal intensity for simpledetection. Preferably 1,000 bases, and more preferably 2,000 bases willsuffice to get good results at a reasonable amount of time. For a reviewof this technique, see Verma et al., (Human Chromosomes: A Manual ofBasic Techniques (Pergamon Press, New York, 1988)).

Reagents for chromosome mapping can be used individually to mark asingle chromosome or a single site on that chromosome, or panels ofreagents can be used for marking multiple sites and/or multiplechromosomes. Reagents corresponding to noncoding regions of the genesactually are preferred for mapping purposes. Coding sequences are morelikely to be conserved within gene families, thus increasing the chanceof cross hybridizations during chromosomal mapping.

Once a sequence has been mapped to a precise chromosomal location, thephysical position of the sequence on the chromosome can be correlatedwith genetic map data. (Such data are found, for example, in V.McKusick, Mendelian Inheritance in Man, available on-line through JohnsHopkins University Welch Medical Library). The relationship betweengenes and disease, mapped to the same chromosomal region, can then beidentified through linkage analysis (co-inheritance of physicallyadjacent genes), described in, e.g., Egeland et al. (1987) Nature,325:783-787.

Moreover, differences in the DNA sequences between individuals affectedand unaffected with a disease associated with the CARD-9, CARD-10, orCARD-11 gene can be determined. If a mutation is observed in some or allof the affected individuals but not in any unaffected individuals, thenthe mutation is likely to be the causative agent of the particulardisease. Comparison of affected and unaffected individuals generallyinvolves first looking for structural alterations in the chromosomessuch as deletions or translocations that are visible from chromosomespreads or detectable using PCR based on that DNA sequence. Ultimately,complete sequencing of genes from several individuals can be performedto confirm the presence of a mutation and to distinguish mutations frompolymorphisms.

A CARD-9, CARD-10, or CARD-11 polypeptide and fragments and sequencesthereof and antibodies specific thereto can be used to map the locationof the gene encoding the polypeptide on a chromosome. This mapping canbe carried out by specifically detecting the presence of the polypeptidein members of a panel of somatic cell hybrids between cells of a firstspecies of animal from which the protein originates and cells from asecond species of animal and then determining which somatic cellhybrid(s) expresses the polypeptide and noting the chromosome(s) fromthe first species of animal that it contains. For examples of thistechnique, see Pajunen et al. (1988) Cytogenet. Cell Genet. 47:37-41 andVan Keuren et al. (1986) Hum. Genet. 74:34-40. Alternatively, thepresence of the CARD-9, CARD-10, or CARD-11 polypeptide in the somaticcell hybrids can be determined by assaying an activity or property ofthe polypeptide, for example, enzymatic activity, as described inBordelon-Riser et al. (1979) Somatic Cell Genetics 5:597-613 andOwerbach et al. (1978) Proc. Natl. Acad. Sci. USA 75:5640-5644.

2. Tissue Typing

The CARD-9, CARD-10, or CARD-11 sequences of the present invention canalso be used to identify individuals from minute biological samples. TheUnited States military, for example, is considering the use ofrestriction fragment length polymorphism (RFLP) for identification ofits personnel. In this technique, an individual's genomic DNA isdigested with one or more restriction enzymes, and probed on a Southernblot to yield unique bands for identification. This method does notsuffer from the current limitations of “Dog Tags” which can be lost,switched, or stolen, making positive identification difficult. Thesequences of the present invention are useful as additional DNA markersfor RFLP (described in U.S. Pat. No. 5,272,057).

Furthermore, the sequences of the present invention can be used toprovide an alternative technique which determines the actualbase-by-base DNA sequence of selected portions of an individual'sgenome. Thus, the CARD-9, CARD-10, or CARD-11 sequences described hereincan be used to prepare two PCR primers from the 5′ and 3′ ends of thesequences. These primers can then be used to amplify an individual's DNAand subsequently sequence it.

Panels of corresponding DNA sequences from individuals, prepared in thismanner, can provide unique individual identifications, as eachindividual will have a unique set of such DNA sequences due to allelicdifferences. The sequences of the present invention can be used toobtain such identification sequences from individuals and from tissue.The CARD-9, CARD-10, or CARD-11 sequences of the invention uniquelyrepresent portions of the human genome. Allelic variation occurs to somedegree in the coding regions of these sequences, and to a greater degreein the noncoding regions. It is estimated that allelic variation betweenindividual humans occurs with a frequency of about once per each 500bases. Each of the sequences described herein can, to some degree, beused as a standard against which DNA from an individual can be comparedfor identification purposes. Because greater numbers of polymorphismsoccur in the noncoding regions, fewer sequences are necessary todifferentiate individuals. The noncoding sequences of SEQ ID NO: 1, SEQID NO: 4, SEQ ID NO: 7, or SEQ ID NO: 10 can comfortably providepositive individual identification with a panel of perhaps 10 to 1,000primers which each yield a noncoding amplified sequence of 100 bases. Ifpredicted coding sequences, such as those in SEQ ID NO: 3, SEQ ID NO: 6,SEQ ID NO: 9, or SEQ ID NO: 12 are used, a more appropriate number ofprimers for positive individual identification would be 500-2,000.

If a panel of reagents from CARD-9, CARD-10, or CARD-11 sequencesdescribed herein is used to generate a unique identification databasefor an individual, those same reagents can later be used to identifytissue from that individual. Using the unique identification database,positive identification of the individual, living or dead, can be madefrom extremely small tissue samples.

3. Use of Partial Sequences in Forensic Biology

DNA-based identification techniques can also be used in forensicbiology. Forensic biology is a scientific field employing genetic typingof biological evidence found at a crime scene as a means for positivelyidentifying, for example, a perpetrator of a crime. To make such anidentification, PCR technology can be used to amplify DNA sequencestaken from very small biological samples such as tissues, e.g., hair orskin, or body fluids, e.g., blood, saliva, or semen found at a crimescene. The amplified sequence can then be compared to a standard,thereby allowing identification of the origin of the biological sample.

The sequences of the present invention can be used to providepolynucleotide reagents, e.g., PCR primers, targeted to specific loci inthe human genome, which can enhance the reliability of DNA-basedforensic identifications by, for example, providing another“identification marker” (i.e. another DNA sequence that is unique to aparticular individual). As mentioned above, actual base sequenceinformation can be used for identification as an accurate alternative topatterns formed by restriction enzyme generated fragments. Sequencestargeted to noncoding regions of SEQ ID NO: 1, SEQ ID NO: 4, SEQ ID NO:7, or SEQ ID NO: 10 are particularly appropriate for this use as greaternumbers of polymorphisms occur in the noncoding regions, making iteasier to differentiate individuals using this technique. Examples ofpolynucleotide reagents include the CARD-9, CARD-10, or CARD-11sequences or portions thereof, e.g., fragments derived from thenoncoding regions of SEQ ID NO: 1, SEQ ID NO: 4, SEQ ID NO: 7, or SEQ IDNO: 10 which have a length of at least 20 or 30 bases.

The sequences described herein can further be used to providepolynucleotide reagents, e.g., labeled or labelable probes which can beused in, for example, an in situ hybridization technique, to identify aspecific tissue, e.g., brain tissue. This can be very useful in caseswhere a forensic pathologist is presented with a tissue of unknownorigin. Panels of such CARD-9, CARD-10, or CARD-11 probes can be used toidentify tissue by species and/or by organ type.

In a similar fashion, these reagents, e.g., CARD-9, CARD-10, or CARD-11primers or probes can be used to screen tissue culture for contamination(i.e., screen for the presence of a mixture of different types of cellsin a culture).

C. Predictive Medicine

The present invention also pertains to the field of predictive medicinein which diagnostic assays, prognostic assays, pharmacogenomics, andmonitoring clinical trials are used for prognostic (predictive) purposesto thereby treat an individual prophylactically. Accordingly, one aspectof the present invention relates to diagnostic assays for determiningCARD-9, CARD-10, or CARD-11 protein and/or nucleic acid expression aswell as CARD-9, CARD-10, or CARD-11 activity, in the context of abiological sample (e.g., blood, serum, cells, tissue) to therebydetermine whether an individual is afflicted with a disease or disorder,or is at risk of developing a disorder, associated with aberrant CARD-9,CARD-10, or CARD-11 expression or activity. The invention also providesfor prognostic (or predictive) assays for determining whether anindividual is at risk of developing a disorder associated with CARD-9,CARD-10, or CARD-11 protein, nucleic acid expression or activity. Forexample, mutations in a CARD-9, CARD-10, or CARD-11 gene can be assayedin a biological sample. Such assays can be used for prognostic orpredictive purpose to thereby prophylactically treat an individual priorto the onset of a disorder characterized by or associated with CARD-9,CARD-10, or CARD-11 protein, nucleic acid expression or activity.

Another aspect of the invention provides methods for determining CARD-9,CARD-10, or CARD-11 protein, nucleic acid expression or CARD-9, CARD-10,or CARD-11 activity in an individual to thereby select appropriatetherapeutic or prophylactic agents for that individual (referred toherein as “pharmacogenomics”). Pharmacogenomics allows for the selectionof agents (e.g., drugs) for therapeutic or prophylactic treatment of anindividual based on the genotype of the individual (e.g., the genotypeof the individual examined to determine the ability of the individual torespond to a particular agent.)

Yet another aspect of the invention pertains to monitoring the influenceof agents (e.g., drugs or other compounds) on the expression or activityof CARD-9, CARD-10, or CARD-11 in clinical trials.

These and other agents are described in further detail in the followingsections.

1. Diagnostic Assays

An exemplary method for detecting the presence or absence of CARD-9,CARD-10, or CARD-11 in a biological sample involves obtaining abiological sample from a test subject and contacting the biologicalsample with a compound or an agent capable of detecting CARD-9, CARD-10,or CARD-11 protein or nucleic acid (e.g., mRNA, genomic DNA) thatencodes CARD-9, CARD-10, or CARD-11 protein such that the presence ofCARD-9, CARD-10, or CARD-11 is detected in the biological sample. Anagent for detecting CARD-9, CARD-10, or CARD-11 mRNA or genomic DNA is alabeled nucleic acid probe capable of hybridizing to CARD-9, CARD-10, orCARD-11 mRNA or genomic DNA. The nucleic acid probe can be, for example,a full-length CARD-9, CARD-10, or CARD-11 nucleic acid, such as thenucleic acid of SEQ ID NO: 1, SEQ ID NO: 3, SEQ ID NO: 4, SEQ ID NO: 6,SEQ ID NO: 7, SEQ ID NO: 9, SEQ ID NO: 10, SEQ ID NO: 12, or a portionthereof, such as an oligonucleotide of at least 15, 30, 50, 100, 250,300, 350, 400, 450, 500, 550, 600, 650, 700, 800, 900, 1000, 1200, 1400,1600, 1800, 2000, 2200, 2400, 2600, 2800, 3000, 3200, 3400, 3600, 3800,4000, 4200, or 4250 nucleotides in length and sufficient to specificallyhybridize under stringent conditions to mRNA or genomic DNA. Othersuitable probes for use in the diagnostic assays of the invention aredescribed herein.

An agent for detecting CARD-9, CARD-10, or CARD-11 protein can be anantibody capable of binding to CARD-9, CARD-10, or CARD-11 protein,preferably an antibody with a detectable label. Antibodies can bepolyclonal, or more preferably, monoclonal. An intact antibody, or afragment thereof (e.g., Fab or F(ab′)2) can be used. The term “labeled”,with regard to the probe or antibody, is intended to encompass directlabeling of the probe or antibody by coupling (i.e., physically linking)a detectable substance to the probe or antibody, as well as indirectlabeling of the probe or antibody by reactivity with another reagentthat is directly labeled. Examples of indirect labeling includedetection of a primary antibody using a fluorescently labeled secondaryantibody and end-labeling of a DNA probe with biotin such that it can bedetected with fluorescently labeled streptavidin. The term “biologicalsample” is intended to include tissues, cells and biological fluidsisolated from a subject, as well as tissues, cells and fluids presentwithin a subject. That is, the detection method of the invention can beused to detect CARD-9, CARD-10, or CARD-11 mRNA, protein, or genomic DNAin a biological sample in vitro as well as in vivo. For example, invitro techniques for detection of CARD-9, CARD-10, or CARD-11 mRNAinclude Northern hybridizations and in situ hybridizations. In vitrotechniques for detection of CARD-9, CARD-10, or CARD-11 protein includeenzyme linked immunosorbent assays (ELISAs), Western blots,immunoprecipitations and immunofluorescence. In vitro techniques fordetection of CARD-9, CARD-10, or CARD-11 genomic DNA include Southernhybridizations. Furthermore, in vivo techniques for detection of CARD-9,CARD-10, or CARD-11 protein include introducing into a subject a labeledanti-CARD-9, CARD-10, or CARD-11 antibody. For example, the antibody canbe labeled with a radioactive marker whose presence and location in asubject can be detected by standard imaging techniques.

In one embodiment, the biological sample contains protein molecules fromthe test subject. Alternatively, the biological sample can contain mRNAmolecules from the test subject or genomic DNA molecules from the testsubject. A biological sample is a peripheral blood leukocyte sampleisolated by conventional means from a subject.

In another embodiment, the methods further involve obtaining a controlbiological sample from a control subject, contacting the control samplewith a compound or agent capable of detecting CARD-9, CARD-10, orCARD-11 protein, mRNA, or genomic DNA, such that the presence of CARD-9,CARD-10, or CARD-11 protein, mRNA or genomic DNA is detected in thebiological sample, and comparing the presence of CARD-9, CARD-10, orCARD-11 protein, mRNA or genomic DNA in the control sample with thepresence of CARD-9, CARD-10, or CARD-11 protein, mRNA or genomic DNA inthe test sample.

The invention also encompasses kits for detecting the presence ofCARD-9, CARD-10, or CARD-11 in a biological sample (a test sample). Suchkits can be used to determine if a subject is suffering from or is atincreased risk of developing a disorder associated with aberrantexpression of CARD-9, CARD-10, or CARD-11 (e.g., an immunologicaldisorder). For example, the kit can comprise a labeled compound or agentcapable of detecting CARD-9, CARD-10, or CARD-11 protein or mRNA in abiological sample and means for determining the amount of CARD-9,CARD-10, or CARD-11 in the sample (e.g., an anti-CARD-9, CARD-10, orCARD-11 antibody or an oligonucleotide probe which binds to DNA encodingCARD-9, CARD-10, or CARD-11, e.g., SEQ ID NO: 1, SEQ ID NO: 3, SEQ IDNO: 4, SEQ ID NO: 6, SEQ ID NO: 7, SEQ ID NO: 9, SEQ ID NO: 10, SEQ IDNO: 12). Kits may also include instruction for observing that the testedsubject is suffering from or is at risk of developing a disorderassociated with aberrant expression of CARD-9, CARD-10, or CARD-10 ifthe amount of CARD-9, CARD-10, or CARD-11 protein or mRNA is above orbelow a normal level.

For antibody-based kits, the kit may comprise, for example: (1) a firstantibody (e.g., attached to a solid support) which binds to CARD-9,CARD-10, or CARD-11 protein; and, optionally, (2) a second, differentantibody which binds to CARD-9, CARD-10, or CARD-11 protein or the firstantibody and is conjugated to a detectable agent. Foroligonucleotide-based kits, the kit may comprise, for example: (1) aoligonucleotide, e.g., a detectably labeled oligonucleotide, whichhybridizes to a CARD-9, CARD-10, or CARD-11 nucleic acid sequence or (2)a pair of primers useful for amplifying a CARD-9, CARD-10, or CARD-11nucleic acid molecule.

The kit may also comprise, e.g., a buffering agent, a preservative, or aprotein stabilizing agent. The kit may also comprise componentsnecessary for detecting the detectable agent (e.g., an enzyme or asubstrate). The kit may also contain a control sample or a series ofcontrol samples which can be assayed and compared to the test samplecontained. Each component of the kit is usually enclosed within anindividual container and all of the various containers are within asingle package along with instructions for observing whether the testedsubject is suffering from or is at risk of developing a disorderassociated with aberrant expression of CARD-9, CARD-10, or CARD-11.

2. Prognostic Assays

The methods described herein can furthermore be utilized as diagnosticor prognostic assays to identify subjects having or at risk ofdeveloping a disease or disorder associated with aberrant CARD-9,CARD-10, or CARD-11 expression or activity. For example, the assaysdescribed herein, such as the preceding diagnostic assays or thefollowing assays, can be utilized to identify a subject having or atrisk of developing a disorder associated with CARD-9, CARD-10, orCARD-11 protein, nucleic acid expression or activity. Alternatively, theprognostic assays can be utilized to identify a subject having or atrisk for developing such a disease or disorder. Thus, the presentinvention provides a method in which a test sample is obtained from asubject and CARD-9, CARD-10, or CARD-11 protein or nucleic acid (e.g.,mRNA, genomic DNA) is detected, wherein the presence of CARD-9, CARD-10,or CARD-11 protein or nucleic acid is diagnostic for a subject having orat risk of developing a disease or disorder associated with aberrantCARD-9, CARD-10, or CARD-11 expression or activity. As used herein, a“test sample” refers to a biological sample obtained from a subject ofinterest. For example, a test sample can be a biological fluid (e.g.,serum), cell sample, or tissue. Furthermore, the prognostic assaysdescribed herein can be used to determine whether a subject can beadministered an agent (e.g., an agonist, antagonist, peptidomimetic,protein, peptide, nucleic acid, small molecule, or other drug candidate)to treat a disease or disorder associated with aberrant CARD-9, CARD-10,or CARD-11 expression or activity. For example, such methods can be usedto determine whether a subject can be effectively treated with aspecific agent or class of agents (e.g., agents of a type which decreaseCARD-9, CARD-10, or CARD-11 activity). Thus, the present inventionprovides methods for determining whether a subject can be effectivelytreated with an agent for a disorder associated with aberrant CARD-9,CARD-10, or CARD-11 expression or activity in which a test sample isobtained and CARD-9, CARD-10, or CARD-11 protein or nucleic acid isdetected (e.g., wherein the presence of CARD-9, CARD-10, or CARD-11protein or nucleic acid is diagnostic for a subject that can beadministered the agent to treat a disorder associated with aberrantCARD-9, CARD-10, or CARD-11 expression or activity).

The methods of the invention can also be used to detect genetic lesionsor mutations in a CARD-9, CARD-10, or CARD-11 gene, thereby determiningif a subject with the lesioned gene is at risk for a disordercharacterized by aberrant cell proliferation and/or differentiation. Inpreferred embodiments, the methods include detecting, in a sample ofcells from the subject, the presence or absence of a genetic lesioncharacterized by at least one of an alteration affecting the integrityof a gene encoding a CARD-9, CARD-10, or CARD-11-protein, or themis-expression of the CARD-9, CARD-10, or CARD-11 gene. For example,such genetic lesions can be detected by ascertaining the existence of atleast one of 1) a deletion of one or more nucleotides from a CARD-9,CARD-10, or CARD-11 gene; 2) an addition of one or more nucleotides to aCARD-9, CARD-10, or CARD-11 gene; 3) a substitution of one or morenucleotides of a CARD-9, CARD-10, or CARD-11 gene; 4) a chromosomalrearrangement of a CARD-9, CARD-10 , or CARD-11 gene; 5) an alterationin the level of a messenger RNA transcript of a CARD-9, CARD-10, orCARD-11 gene; 6) aberrant modification of a CARD-9, CARD-10, or CARD-11gene, such as of the methylation pattern of the genomic DNA; 7) thepresence of a non-wild type splicing pattern of a messenger RNAtranscript of a CARD-9, CARD-10, or CARD-11 gene (e.g., caused by amutation in a splice donor or splice acceptor site); 8) a non-wild typelevel of a CARD-9, CARD-10, or CARD-11-protein; 9) allelic loss of aCARD-9, CARD-10, or CARD-11 gene; and 10) inappropriatepost-translational modification of a CARD-9, CARD-10, orCARD-11-protein. As described herein, there are a large number of assaytechniques known in the art which can be used for detecting lesions in aCARD-9, CARD-10, or CARD-11 gene. A biological sample is a peripheralblood leukocyte sample isolated by conventional means from a subject.

In certain embodiments, detection of the lesion involves the use of aprobe/primer in a polymerase chain reaction (PCR) (see, e.g., U.S. Pat.Nos. 4,683,195 and 4,683,202), such as anchor PCR or RACE PCR, or,alternatively, in a ligation chain reaction (LCR) (see, e.g., Landegranet al. (1988) Science 241:1077-1080; and Nakazawa et al. (1994) Proc.Natl. Acad. Sci. USA 91:360-364), the latter of which can beparticularly useful for detecting point mutations in the CARD-9,CARD-10, or CARD-11 gene (see, e.g., Abravaya et al. (1995) NucleicAcids Res. 23:675-682). This method can include the steps of collectinga sample of cells from a patient, isolating nucleic acid (e.g., genomic,mRNA or both) from the cells of the sample, contacting the nucleic acidsample with one or more primers which specifically hybridize to aCARD-9, CARD-10, or CARD-11 gene under conditions such thathybridization and amplification of the CARD-9, CARD-10, or CARD-11-gene(if present) occurs, and detecting the presence or absence of anamplification product, or detecting the size of the amplificationproduct and comparing the length to a control sample. It is anticipatedthat PCR and/or LCR may be desirable to use as a preliminaryamplification step in conjunction with any of the techniques used fordetecting mutations described herein.

Alternative amplification methods include: self sustained sequencereplication (Guatelli et al. (1990) Proc. Natl. Acad. Sci. USA87:1874-1878), transcriptional amplification system (Kwoh, et al. (1989)Proc. Natl. Acad. Sci. USA 86:1173-1177), Q-Beta Replicase (Lizardi etal. (1988) Bio/Technology 6:1197), or any other nucleic acidamplification method, followed by the detection of the amplifiedmolecules using techniques well known to those of skill in the art.These detection schemes are especially useful for the detection ofnucleic acid molecules if such molecules are present in very lownumbers.

In an alternative embodiment, mutations in a CARD-9, CARD-10, or CARD-11gene from a sample cell can be identified by alterations in restrictionenzyme cleavage patterns. For example, sample and control DNA isisolated, amplified (optionally), digested with one or more restrictionendonucleases, and fragment length sizes are determined by gelelectrophoresis and compared. Differences in fragment length sizesbetween sample and control DNA indicates mutations in the sample DNA.Moreover, the use of sequence specific ribozymes (see, e.g., U.S. Pat.No. 5,498,531) can be used to score for the presence of specificmutations by development or loss of a ribozyme cleavage site.

In other embodiments, genetic mutations in CARD-9, CARD-10, or CARD-11can be identified by hybridizing a sample and control nucleic acids,e.g., DNA or RNA, to high density arrays containing hundreds orthousands of oligonucleotides probes (Cronin et al. (1996) HumanMutation 7:244-255; Kozal et al. (1996) Nature Medicine 2:753-759). Forexample, genetic mutations in CARD-9, CARD-10, or CARD-11 can beidentified in two-dimensional arrays containing light-generated DNAprobes as described in Cronin et al. supra. Briefly, a firsthybridization array of probes can be used to scan through long stretchesof DNA in a sample and control to identify base changes between thesequences by making linear arrays of sequential overlapping probes. Thisstep allows the identification of point mutations. This step is followedby a second hybridization array that allows the characterization ofspecific mutations by using smaller, specialized probe arrayscomplementary to all variants or mutations detected. Each mutation arrayis composed of parallel probe sets, one complementary to the wild-typegene and the other complementary to the mutant gene.

In yet another embodiment, any of a variety of sequencing reactionsknown in the art can be used to directly sequence the CARD-9, CARD-10,or CARD-11 gene and detect mutations by comparing the sequence of thesample CARD-9, CARD-10, or CARD-11 with the corresponding wild-type(control) sequence. Examples of sequencing reactions include those basedon techniques developed by Maxam and Gilbert ((1977) Proc. Natl. Acad.Sci. USA 74:560) or Sanger ((1977) Proc. Natl. Acad. Sci. USA 74:5463).It is also contemplated that any of a variety of automated sequencingprocedures can be utilized when performing the diagnostic assays ((1995)Bio/Techniques 19:448), including sequencing by mass spectrometry (see,e.g., PCT Publication No. WO 94/16101; Cohen et al. (1996) Adv.Chromatogr. 36:127-162; and Griffin et al. (1993) Appl. Biochem.Biotechnol. 38:147-159).

Other methods for detecting mutations in the CARD-9, CARD-10, or CARD-11gene include methods in which protection from cleavage agents is used todetect mismatched bases in RNA/RNA or RNA/DNA heteroduplexes (Myers etal. (1985) Science 230:1242). In general, the art technique of “mismatchcleavage” starts by providing heteroduplexes of formed by hybridizing(labeled) RNA or DNA containing the wild-type CARD-9, CARD-10, orCARD-11 sequence with potentially mutant RNA or DNA obtained from atissue sample. The double-stranded duplexes are treated with an agentwhich cleaves single-stranded regions of the duplex such as which willexist due to basepair mismatches between the control and sample strands.For instance, RNA/DNA duplexes can be treated with RNase and DNA/DNAhybrids treated with S1 nuclease to enzymatically digesting themismatched regions. In other embodiments, either DNA/DNA or RNA/DNAduplexes can be treated with hydroxylamine or osmium tetroxide and withpiperidine in order to digest mismatched regions. After digestion of themismatched regions, the resulting material is then separated by size ondenaturing polyacrylamide gels to determine the site of mutation. See,e.g., Cotton et al (1988) Proc. Natl Acad Sci USA 85:4397; Saleeba et al(1992) Methods Enzymol. 217:286-295. In an embodiment, the control DNAor RNA can be labeled for detection.

In still another embodiment, the mismatch cleavage reaction employs oneor more proteins that recognize mismatched base pairs in double-strandedDNA (so called “DNA mismatch repair” enzymes) in defined systems fordetecting and mapping point mutations in CARD-9, CARD-10, or CARD-11cDNAs obtained from samples of cells. For example, the mutY enzyme of E.coli cleaves A at G/A mismatches and the thymidine DNA glycosylase fromHeLa cells cleaves T at G/T mismatches (Hsu et al. (1994) Carcinogenesis15:1657-1662). According to an exemplary embodiment, a probe based on aCARD-9, CARD-10, or CARD-11 sequence, e.g., a wild-type CARD-9, CARD-10,or CARD-11 sequence, is hybridized to a cDNA or other DNA product from atest cell(s). The duplex is treated with a DNA mismatch repair enzyme,and the cleavage products, if any, can be detected from electrophoresisprotocols or the like. See, e.g., U.S. Pat. No. 5,459,039.

In other embodiments, alterations in electrophoretic mobility will beused to identify mutations in CARD-9, CARD-10, or CARD-11 genes. Forexample, single strand conformation polymorphism (SSCP) may be used todetect differences in electrophoretic mobility between mutant and wildtype nucleic acids (Orita et al. (1989) Proc Natl. Acad. Sci USA:86:2766, see also Cotton (1993) Mutat. Res. 285:125-144; and Hayashi(1992) Genet Anal Tech Appl 9:73-79). Single-stranded DNA fragments ofsample and control CARD-9, CARD-10, or CARD-11 nucleic acids will bedenatured and allowed to renature. The secondary structure ofsingle-stranded nucleic acids varies according to sequence, theresulting alteration in electrophoretic mobility enables the detectionof even a single base change. The DNA fragments may be labeled ordetected with labeled probes. The sensitivity of the assay may beenhanced by using RNA (rather than DNA), in which the secondarystructure is more sensitive to a change in sequence. In an embodiment,the subject method utilizes heteroduplex analysis to separate doublestranded heteroduplex molecules on the basis of changes inelectrophoretic mobility (Keen et al. (1991) Trends Genet 7:5).

In yet another embodiment, the movement of mutant or wild-type fragmentsin polyacrylamide gels containing a gradient of denaturant is assayedusing denaturing gradient gel electrophoresis (DGGE) (Myers et al.(1985) Nature 313:495). When DGGE is used as the method of analysis, DNAwill be modified to insure that it does not completely denature, forexample by adding a GC clamp of approximately 40 bp of high-meltingGC-rich DNA by PCR. In a further embodiment, a temperature gradient isused in place of a denaturing gradient to identify differences in themobility of control and sample DNA (Rosenbaum and Reissner (1987)Biophys Chem 265:12753).

Examples of other techniques for detecting point mutations include, butare not limited to, selective oligonucleotide hybridization, selectiveamplification, or selective primer extension. For example,oligonucleotide primers may be prepared in which the known mutation isplaced centrally and then hybridized to target DNA under conditionswhich permit hybridization only if a perfect match is found (Saiki etal. (1986) Nature 324:163); Saiki et al. (1989) Proc. Natl Acad. Sci USA86:6230). Such allele specific oligonucleotides are hybridized to PCRamplified target DNA or a number of different mutations when theoligonucleotides are attached to the hybridizing membrane and hybridizedwith labeled target DNA.

Alternatively, allele specific amplification technology which depends onselective PCR amplification may be used in conjunction with the instantinvention. Oligonucleotides used as primers for specific amplificationmay carry the mutation of interest in the center of the molecule (sothat amplification depends on differential hybridization) (Gibbs et al.(1989) Nucleic Acids Res. 17:2437-2448) or at the extreme 3′ end of oneprimer where, under appropriate conditions, mismatch can prevent, orreduce polymerase extension (Prossner (1993) Tibtech 11:238). Inaddition, it may be desirable to introduce a novel restriction site inthe region of the mutation to create cleavage-based detection (Gaspariniet al. (1992) Mol. Cell Probes 6:1). It is anticipated that in certainembodiments amplification may also be performed using Taq ligase foramplification (Barany (1991) Proc. Natl. Acad. Sci USA 88:189). In suchcases, ligation will occur only if there is a perfect match at the 3′end of the 5′ sequence making it possible to detect the presence of aknown mutation at a specific site by looking for the presence or absenceof amplification.

The methods described herein may be performed, for example, by utilizingpre-packaged diagnostic kits comprising at least one probe nucleic acidor antibody reagent described herein, which may be conveniently used,e.g., in clinical settings to diagnose patients exhibiting symptoms orfamily history of a disease or illness involving a CARD-9, CARD-10, orCARD-11 gene.

Furthermore, any cell type or tissue, preferably peripheral bloodleukocytes, in which CARD-9, CARD-10, or CARD-11 is expressed may beutilized in the prognostic assays described herein.

3. Pharmacogenomics

Agents, or modulators which have a stimulatory or inhibitory effect onCARD-9, CARD-10, or CARD-11 activity (e.g., CARD-9, CARD-10, or CARD-11gene expression) as identified by a screening assay described herein canbe administered to individuals to treat (prophylactically ortherapeutically) disorders (e.g., an immunological disorder) associatedwith aberrant CARD-9, CARD-10, or CARD-11 activity. In conjunction withsuch treatment, the pharmacogenomics (i.e., the study of therelationship between an individual's genotype and that individual'sresponse to a foreign compound or drug) of the individual may beconsidered. Differences in metabolism of therapeutics can lead to severetoxicity or therapeutic failure by altering the relation between doseand blood concentration of the pharmacologically active drug. Thus, thepharmacogenomics of the individual permits the selection of effectiveagents (e.g., drugs) for prophylactic or therapeutic treatments based ona consideration of the individual's genotype. Such pharmacogenomics canfurther be used to determine appropriate dosages and therapeuticregimens. Accordingly, the activity of CARD-9, CARD-10, or CARD-11protein, expression of CARD-9, CARD-10, or CARD-11 nucleic acid, ormutation content of CARD-9, CARD-10, or CARD-11 genes in an individualcan be determined to thereby select appropriate agent(s) for therapeuticor prophylactic treatment of the individual.

Pharmacogenomics deals with clinically significant hereditary variationsin the response to drugs due to altered drug disposition and abnormalaction in affected persons. See, e.g., Linder (1997) Clin. Chem.43(2):254-266. In general, two types of pharmacogenetic conditions canbe differentiated. Genetic conditions transmitted as a single factoraltering the way drugs act on the body (altered drug action) or geneticconditions transmitted as single factors altering the way the body actson drugs (altered drug metabolism). These pharmacogenetic conditions canoccur either as rare defects or as polymorphisms. For example,glucose-6-phosphate dehydrogenase deficiency (G6PD) is a commoninherited enzymopathy in which the main clinical complication ishaemolysis after ingestion of oxidant drugs (anti-malarials,sulfonamides, analgesics, nitrofurans) and consumption of fava beans.

As an illustrative embodiment, the activity of drug metabolizing enzymesis a major determinant of both the intensity and duration of drugaction. The discovery of genetic polymorphisms of drug metabolizingenzymes (e.g., N-acetyltransferase 2 (NAT 2) and cytochrome P450 enzymesCYP2D6 and CYP2C 19) has provided an explanation as to why some patientsdo not obtain the expected drug effects or show exaggerated drugresponse and serious toxicity after taking the standard and safe dose ofa drug. These polymorphisms are expressed in two phenotypes in thepopulation, the extensive metabolizer (EM) and poor metabolizer (PM).The prevalence of PM is different among different populations. Forexample, the gene coding for CYP2D6 is highly polymorphic and severalmutations have been identified in PM, which all lead to the absence offunctional CYP2D6. Poor metabolizers of CYP2D6 and CYP2C19 quitefrequently experience exaggerated drug response and side effects whenthey receive standard doses. If a metabolite is the active therapeuticmoiety, PM exhibit no therapeutic response, as demonstrated for theanalgesic effect of codeine mediated by its CYP2D6-formed metabolitemorphine. The other extreme are the so-called ultra-rapid metabolizerswho do not respond to standard doses. Recently, the molecular basis ofultra-rapid metabolism has been identified to be due to CYP2D6 geneamplification.

Thus, the activity of CARD-9, CARD-10, or CARD-11 protein, expression ofCARD-9, CARD-10, or CARD-11 nucleic acid, or mutation content of CARD-9,CARD-10, or CARD-11 genes in an individual can be determined to therebyselect appropriate agent(s) for therapeutic or prophylactic treatment ofthe individual. In addition, pharmacogenetic studies can be used toapply genotyping of polymorphic alleles encoding drug-metabolizingenzymes to the identification of an individual's drug responsivenessphenotype. This knowledge, when applied to dosing or drug selection, canavoid adverse reactions or therapeutic failure and thus enhancetherapeutic or prophylactic efficiency when treating a subject with aCARD-9, CARD-10, or CARD-11 modulator, such as a modulator identified byone of the exemplary screening assays described herein.

4. Monitoring of Effects During Clinical Trials

Monitoring the influence of agents (e.g., drugs, compounds) on theexpression or activity of CARD-9, CARD-10, or CARD-11 (e.g., the abilityto modulate aberrant cell proliferation and/or differentiation) can beapplied not only in basic drug screening, but also in clinical trials.For example, the effectiveness of an agent determined by a screeningassay as described herein to increase CARD-9, CARD-10, or CARD-11 geneexpression, protein levels, or upregulate CARD-9, CARD-10, or CARD-11activity, can be monitored in clinical trails of subjects exhibitingdecreased CARD-9, CARD-10, or CARD-11 gene expression, protein levels,or downregulated CARD-9, CARD-10, or CARD-11 activity. Alternatively,the effectiveness of an agent determined by a screening assay todecrease CARD-9, CARD-10, or CARD-11 gene expression, protein levels, ordownregulated CARD-9, CARD-10, or CARD-11 activity, can be monitored inclinical trials of subjects exhibiting increased CARD-9, CARD-10, orCARD-11 gene expression, protein levels, or upregulated CARD-9, CARD-10,or CARD-11 activity. In such clinical trials, the expression or activityof CARD-9, CARD-10, or CARD-11 and, preferably, other genes that havebeen implicated in, for example, a cellular proliferation disorder canbe used as a “read out” or markers of the immune responsiveness of aparticular cell.

For example, and not by way of limitation, genes, including CARD-9,CARD-10, or CARD-11, that are modulated in cells by treatment with anagent (e.g., compound, drug or small molecule) which modulates CARD-9,CARD-10, or CARD-11 activity (e.g., identified in a screening assay asdescribed herein) can be identified. Thus, to study the effect of agentson cellular proliferation disorders, for example, in a clinical trial,cells can be isolated and RNA prepared and analyzed for the levels ofexpression of CARD-9, CARD-10, or CARD-11 and other genes implicated inthe disorder. The levels of gene expression (i.e., a gene expressionpattern) can be quantified by Northern blot analysis or RT-PCR, asdescribed herein, or alternatively by measuring the amount of proteinproduced, by one of the methods as described herein, or by measuring thelevels of activity of CARD-9, CARD-10, or CARD-11 or other genes. Inthis way, the gene expression pattern can serve as a marker, indicativeof the physiological response of the cells to the agent. Accordingly,this response state may be determined before, and at various pointsduring, treatment of the individual with the agent.

In an embodiment, the present invention provides a method for monitoringthe effectiveness of treatment of a subject with an agent (e.g., anagonist, antagonist, peptidomimetic, protein, peptide, nucleic acid,small molecule, or other drug candidate identified by the screeningassays described herein) comprising the steps of (i) obtaining apre-administration sample from a subject prior to administration of theagent; (ii) detecting the level of expression of a CARD-9, CARD-10, orCARD-11 protein, mRNA, or genomic DNA in the preadministration sample;(iii) obtaining one or more post-administration samples from thesubject; (iv) detecting the level of expression or activity of theCARD-9, CARD-10, or CARD-11 protein, mRNA, or genomic DNA in thepost-administration samples; (v) comparing the level of expression oractivity of the CARD-9, CARD-10, or CARD-11 protein, mRNA, or genomicDNA in the pre-administration sample with the CARD-9, CARD-10, orCARD-11 protein, mRNA, or genomic DNA in the post administration sampleor samples; and (vi) altering the administration of the agent to thesubject accordingly. For example, increased administration of the agentmay be desirable to increase the expression or activity of CARD-9,CARD-10, or CARD-11 to higher levels than detected, i.e., to increasethe effectiveness of the agent. Alternatively, decreased administrationof the agent may be desirable to decrease expression or activity ofCARD-9, CARD-10, or CARD-11 to lower levels than detected, i.e., todecrease the effectiveness of the agent.

5. Transcriptional Profiling

The CARD-9, CARD-10, or CARD-11 nucleic acid molecules described herein,including small oligonucleotides, can be used in transcriptionallyprofiling. For example, these nucleic acids can be used to examine theexpression of CARD-9, CARD-10, or CARD-11 in normal tissue or cells andin tissue or cells subject to a disease state, e.g., tissue or cellsderived from a patient having a disease of interest or cultured cellswhich model or reflect a disease state of interest, e.g., cells of acultured tumor cell line. By measuring expression of CARD-9, CARD-10, orCARD-11, together or individually, a profile of expression in normal anddisease states can be developed. This profile can be used diagnosticallyand to examine the effectiveness of a therapeutic regime.

C. Methods of Treatment

The present invention provides for both prophylactic and therapeuticmethods of treating a subject at risk of (or susceptible to) a disorderor having a disorder associated with aberrant CARD-9, CARD-10, orCARD-11 expression or activity, examples of which are provided herein.

1. Prophylactic Methods

In one aspect, the invention provides a method for preventing in asubject, a disease or condition associated with an aberrant CARD-9,CARD-10, or CARD-11 expression or activity, by administering to thesubject an agent which modulates CARD-9, CARD-10, or CARD-11 expressionor at least one CARD-9, CARD-10, or CARD-11 activity. Subjects at riskfor a disease which is caused or contributed to by aberrant CARD-9,CARD-10, or CARD-11 expression or activity can be identified by, forexample, any or a combination of diagnostic or prognostic assays asdescribed herein. Administration of a prophylactic agent can occur priorto the manifestation of symptoms characteristic of the CARD-9, CARD-10,or CARD-11 aberrancy, such that a disease or disorder is prevented or,alternatively, delayed in its progression. Depending on the type ofCARD-9, CARD-10, or CARD-11 aberrancy, for example, a CARD-9, CARD-10,or CARD-11 agonist or CARD-9, CARD-10, or CARD-11 antagonist agent canbe used for treating the subject. The appropriate agent can bedetermined based on screening assays described herein.

2. Therapeutic Methods

Another aspect of the invention pertains to methods of modulatingCARD-9, CARD-10, or CARD-11 expression or activity for therapeuticpurposes. The modulatory method of the invention involves contacting acell with an agent that modulates one or more of the activities ofCARD-9, CARD-10, or CARD-11 protein activity associated with the cell.An agent that modulates CARD-9, CARD-10, or CARD-11 protein activity canbe an agent as described herein, such as a nucleic acid or a protein, anaturally-occurring cognate ligand of a CARD-9, CARD-10, or CARD-11protein, a peptide, a CARD-9, CARD-10, or CARD-11 peptidomimetic, orother small molecule. In one embodiment, the agent stimulates one ormore of the biological activities of CARD-9, CARD-10, or CARD-11protein. Examples of such stimulatory agents include active CARD-9,CARD-10, or CARD-11 protein and a nucleic acid molecule encoding CARD-9,CARD-10, or CARD-11 that has been introduced into the cell. In anotherembodiment, the agent inhibits one or more of the biological activitiesof CARD-9, CARD-10, or CARD-11 protein. Examples of such inhibitoryagents include antisense CARD-9, CARD-10, or CARD-11 nucleic acidmolecules and anti-CARD-9, CARD-10, or CARD-11 antibodies. Thesemodulatory methods can be performed in vitro (e.g., by culturing thecell with the agent) or, alternatively, in vivo (e.g., by administeringthe agent to a subject). As such, the present invention provides methodsof treating an individual afflicted with a disease or disordercharacterized by aberrant expression or activity of a CARD-9, CARD-10,or CARD-11 protein or nucleic acid molecule or a disorder related toCARD-9, CARD-10, or CARD-11 expression or activity. In one embodiment,the method involves administering an agent (e.g., an agent identified bya screening assay described herein), or combination of agents thatmodulates (e.g., upregulates or downregulates) CARD-9, CARD-10, orCARD-11 expression or activity. In another embodiment, the methodinvolves administering a CARD-9, CARD-10, or CARD-11 protein or nucleicacid molecule as therapy to compensate for reduced or aberrant CARD-9,CARD-10, or CARD-11 expression or activity. Stimulation of CARD-9,CARD-10, or CARD-11 activity is desirable in situations in which CARD-9,CARD-10, or CARD-11 is abnormally downregulated and/or in whichincreased CARD-9, CARD-10, or CARD-11 activity is likely to have abeneficial effect. Conversely, inhibition of CARD-9, CARD-10, or CARD-11activity is desirable in situations in which CARD-9, CARD-10, or CARD-11is abnormally upregulated, e.g., in myocardial infarction, and/or inwhich decreased CARD-9, CARD-10, or CARD-11 activity is likely to have abeneficial effect.

The contents of all references, patents and published patentapplications cited throughout this application are hereby incorporatedby reference.

Equivalents

Those skilled in the art will recognize, or be able to ascertain usingno more than routine experimentation, many equivalents to the specificembodiments of the invention described herein. Such equivalents areintended to be encompassed by the following claims.

1. An isolated polypeptide selected from the group consisting of: a) a polypeptide comprising a fragment of the amino acid sequence of SEQ ID NO: 11, wherein the fragment comprises the CARD domain of residues 6-112 of SEQ ID NO: 11, wherein the fragment binds Bcl-10; b) a polypeptide comprising a variant of the amino acid sequence of SEQ ID NO: 11, wherein the polypeptide is encoded by a nucleic acid molecule which hybridizes to a nucleic acid molecule consisting of the entire complement of SEQ ID NO: 12 under conditions of incubation at 45° C. in 6.0X SSC followed by washing in 0.2X SSC/0.1% SDS at 65° C., wherein the variant binds Bcl-10 and comprises a CARD domain; c) a polypeptide which is encoded by a nucleic acid molecule comprising a nucleotide sequence which is at least 95% identical to SEQ ID NO: 10, 12, wherein the polypeptide binds Bcl-10 and comprises a CARD domain; and d) a polypeptide comprising an amino acid sequence that is at least 95% identical to SEQ ID NO: 11, wherein the polypeptide binds Bcl-10 and comprises a CARD domain.
 2. The isolated polypeptide of claim 1 comprising the amino acid sequence of SEQ ID NO:
 11. 3. The polypeptide of claim 1 further comprising heterologous amino acid sequences.
 4. The polypeptide of claim 1, wherein the fragment further comprises amino acids 635-1147 of SEQ ID NO:
 11. 5. The polypeptide of claim 1, wherein the polypeptide comprises an amino acid sequence which is at least 98% identical to the amino acid sequence of SEQ ID NO:
 11. 6. An isolated polypeptide consisting of the amino acid sequence of SEQ ID NO:
 11. 7. The isolated polypeptide of claim 1, wherein the polypeptide of claim 1 b), c) or d) comprises the CARD domain of residues 6-112 of SEQ ID NO:
 11. 8. The isolated polypeptide of claim 5, wherein the polypeptide comprises the CARD domain of residues 6-112 of SEQ ID) NO:
 11. 9. The isolated polypeptide of claim 1, wherein the polypeptide further comprises the coiled coil domain of residues 130-431 of SEQ ID NO:
 11. 